Betacoronavirus mRNA vaccine

ABSTRACT

The disclosure relates to respiratory virus ribonucleic acid (RNA) vaccines and combination vaccines, as well as methods of using the vaccines and compositions comprising the vaccines.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/805,587,filed Feb. 28, 2020, now U.S. Pat. No. 10,702,600, which is acontinuation of U.S. application Ser. No. 16/368,270, filed Mar. 28,2019, now U.S. Pat. No. 10,702,599, which is a continuation of Ser. No.16/040,981, filed Jul. 20, 2018, now U.S. Pat. No. 10,272,150, which isa continuation of U.S. application Ser. No. 15/674,599, filed Aug. 11,2017, now U.S. Pat. No. 10,064,934, which is a continuation ofInternational application number PCT/US2016/058327, filed Oct. 21, 2016,which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/244,802, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,297, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/244,946, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,362, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/244,813, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,394, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/244,837, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,483, filed Oct. 28, 2015, and U.S. provisionalapplication No. 62/245,031, filed Oct. 22, 2015, each of which isincorporated by reference herein in its entirety.

BACKGROUND

Respiratory disease is a medical term that encompasses pathologicalconditions affecting the organs and tissues that make gas exchangepossible in higher organisms, and includes conditions of the upperrespiratory tract, trachea, bronchi, bronchioles, alveoli, pleura andpleural cavity, and the nerves and muscles of breathing. Respiratorydiseases range from mild and self-limiting, such as the common cold, tolife-threatening entities like bacterial pneumonia, pulmonary embolism,acute asthma and lung cancer. Respiratory disease is a common andsignificant cause of illness and death around the world. In the US,approximately 1 billion “common colds” occur each year. Respiratoryconditions are among the most frequent reasons for hospital stays amongchildren.

The human Metapneumovirus (hMPV) is a negative-sense, single-strandedRNA virus of the genus Pneumovirinae and of the family Paramyxoviridaeand is closely related to the avian Metapneumovirus (AMPV) subgroup C.It was isolated for the first time in 2001 in the Netherlands by usingthe RAP-PCR (RNA arbitrarily primed PCR) technique for identification ofunknown viruses growing in cultured cells. hPMV is second only to RSV asan important cause of viral lower respiratory tract illness (LRI) inyoung children. The seasonal epidemiology of hMPV appears to be similarto that of RSV, but the incidence of infection and illness appears to besubstantially lower.

Parainfluenza virus type 3 (PIV3), like hMPV, is also a negative-sense,single-stranded sense RNA virus of the genus Pneumovirinae and of thefamily Paramyxoviridae and is a major cause of ubiquitous acuterespiratory infections of infancy and early childhood. Its incidencepeaks around 4-12 months of age, and the virus is responsible for 3-10%of hospitalizations, mainly for bronchiolitis and pneumonia. PIV3 can befatal, and in some instances is associated with neurologic diseases,such as febrile seizures. It can also result in airway remodeling, asignificant cause of morbidity. In developing regions of the world,infants and young children are at the highest risk of mortality, eitherfrom primary PIV3 viral infection or a secondary consequences, such asbacterial infections. Human parainfluenza viruses (hPIV) types 1, 2 and3 (hPIV1, hPIV2 and hPIV3, respectively), also like hMPV, are secondonly to RSV as important causes of viral LRI in young children.

RSV, too, is a negative-sense, single-stranded RNA virus of the genusPneumovirinae and of the family Paramyxoviridae. Symptoms in adultstypically resemble a sinus infection or the common cold, although theinfection may be asymptomatic. In older adults (e.g., >60 years), RSVinfection may progress to bronchiolitis or pneumonia. Symptoms inchildren are often more severe, including bronchiolitis and pneumonia.It is estimated that in the United States, most children are infectedwith RSV by the age of three. The RSV virion consists of an internalnucleocapsid comprised of the viral RNA bound to nucleoprotein (N),phosphoprotein (P), and large polymerase protein (L). The nucleocapsidis surrounded by matrix protein (M) and is encapsulated by a lipidbilayer into which the viral fusion (F) and attachment (G) proteins aswell as the small hydrophobic protein (SH) are incorporated. The viralgenome also encodes two nonstructural proteins (NS1 and NS2), whichinhibit type I interferon activity as well as the M-2 protein.

The continuing health problems associated with hMPV, PIV3 and RSV are ofconcern internationally, reinforcing the importance of developingeffective and safe vaccine candidates against these virus.

Despite decades of research, no vaccines currently exist (Sato andWright, Pediatr. Infect. Dis. J. 2008; 27(10 Suppl):S123-5). Recombinanttechnology, however, has been used to target the formation of vaccinesfor hPIV-1, 2 and 3 serotypes, for example, and has taken the form ofseveral live-attenuated intranasal vaccines. Two vaccines in particularwere found to be immunogenic and well tolerated against hPIV-3 in phaseI trials. hPIV1 and hPIV2 vaccine candidates remain less advanced(Durbin and Karron, Clinical infectious diseases: an officialpublication of the Infectious Diseases Society of America 2003;37(12):1668-77).

Measles virus (MeV), like hMPV, PIV3 and RSV, is a negative-sense,single-stranded RNA virus that is the cause of measles, an infection ofthe respiratory system. MeV is of the genus Morbillivirus within thefamily Paramyxoviridae. Humans are the natural hosts of the virus; noanimal reservoirs are known to exist. Symptoms of measles include fever,cough, runny nose, red eyes and a generalized, maculopapular,erythematous rash. The virus is highly contagious and is spread bycoughing

In additional to hMPV, PIV, RSV and MeV, Betacoronaviruses are known tocause respiratory illnesses. Betacoronaviruses (BetaCoVs) are one offour genera of coronaviruses of the subfamily Coronavirinae in thefamily Coronaviridae, of the order Nidovirales. They are enveloped,positive-sense, single-stranded RNA viruses of zoonotic origin. Thecoronavirus genera are each composed of varying viral lineages, with theBetacoronavirus genus containing four such lineages. The BetaCoVs of thegreatest clinical importance concerning humans are OC43 and HKU1 of theA lineage, SARS-CoV of the B lineage, and MERS-CoV of the C lineage.MERS-CoV is the first Betacoronavirus belonging to lineage C that isknown to infect humans.

The Middle East respiratory syndrome coronavirus (MERS-CoV), or EMC/2012(HCoV-EMC/2012), initially referred to as novel coronavirus 2012 orsimply novel coronavirus, was first reported in 2012 after genomesequencing of a virus isolated from sputum samples from a person whofell ill during a 2012 outbreak of a new flu. As of July 2015, MERS-CoVcases have been reported in over 21 countries. The outbreaks of MERS-CoVhave raised serious concerns world-wide, reinforcing the importance ofdeveloping effective and safe vaccine candidates against MERS-CoV.

Severe acute respiratory syndrome (SARS) emerged in China in 2002 andspread to other countries before brought under control. Because of aconcern for reemergence or a deliberate release of the SARS coronavirus,vaccine development was initiated.

Deoxyribonucleic acid (DNA) vaccination is one technique used tostimulate humoral and cellular immune responses to foreign antigens,such as hMPV antigens and/or PIV antigens and/or RSV antigens. Thedirect injection of genetically engineered DNA (e.g., naked plasmid DNA)into a living host results in a small number of its cells directlyproducing an antigen, resulting in a protective immunological response.With this technique, however, comes potential problems, including thepossibility of insertional mutagenesis, which could lead to theactivation of oncogenes or the inhibition of tumor suppressor genes.

SUMMARY

Provided herein are ribonucleic acid (RNA) vaccines that build on theknowledge that RNA (e.g., messenger RNA (mRNA)) can safely direct thebody's cellular machinery to produce nearly any protein of interest,from native proteins to antibodies and other entirely novel proteinconstructs that can have therapeutic activity inside and outside ofcells. The RNA (e.g., mRNA) vaccines of the present disclosure may beused to induce a balanced immune response against hMPV, PIV, RSV, MeV,and/or BetaCoV (e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E,HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1), or any combination of twoor more of the foregoing viruses, comprising both cellular and humoralimmunity, without risking the possibility of insertional mutagenesis,for example. hMPV, PIV, RSV, MeV, BetaCoV (e.g., MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1) andcombinations thereof are referred to herein as “respiratory viruses.”Thus, the term “respiratory virus RNA vaccines” encompasses hMPV RNAvaccines, PIV RNA vaccines, RSV RNA vaccines, MeV RNA vaccines, BetaCoVRNA vaccines, and any combination of two or more of hMPV RNA vaccines,PIV RNA vaccines, RSV RNA vaccines, MeV RNA vaccines, and BetaCoV RNAvaccines.

The RNA (e.g., mRNA) vaccines may be utilized in various settingsdepending on the prevalence of the infection or the degree or level ofunmet medical need. The RNA (e.g. mRNA) vaccines may be utilized totreat and/or prevent a hMPV, PIV, RSV, MeV, a BetaCoV (e.g., MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1),or any combination of two or more of the foregoing viruses, of variousgenotypes, strains, and isolates. The RNA (e.g., mRNA) vaccines havesuperior properties in that they produce much larger antibody titers andproduce responses earlier than commercially available anti-viraltherapeutic treatments. While not wishing to be bound by theory, it isbelieved that the RNA (e.g., mRNA) vaccines, as mRNA polynucleotides,are better designed to produce the appropriate protein conformation upontranslation as the RNA (e.g., mRNA) vaccines co-opt natural cellularmachinery. Unlike traditional vaccines, which are manufactured ex vivoand may trigger unwanted cellular responses, RNA (e.g., mRNA) vaccinesare presented to the cellular system in a more native fashion.

In some aspects the invention is a respiratory virus vaccine, comprisingat least one RNA polynucleotide having an open reading frame encoding atleast one respiratory virus antigenic polypeptide, formulated in acationic lipid nanoparticle.

Surprisingly, in some aspects it has also been shown that efficacy ofmRNA vaccines can be significantly enhanced when combined with aflagellin adjuvant, in particular, when one or more antigen-encodingmRNAs is combined with an mRNA encoding flagellin.

RNA (e.g., mRNA) vaccines combined with the flagellin adjuvant (e.g.,mRNA-encoded flagellin adjuvant) have superior properties in that theymay produce much larger antibody titers and produce responses earlierthan commercially available vaccine formulations. While not wishing tobe bound by theory, it is believed that the RNA (e.g., mRNA) vaccines,for example, as mRNA polynucleotides, are better designed to produce theappropriate protein conformation upon translation, for both the antigenand the adjuvant, as the RNA (e.g., mRNA) vaccines co-opt naturalcellular machinery. Unlike traditional vaccines, which are manufacturedex vivo and may trigger unwanted cellular responses, RNA (e.g., mRNA)vaccines are presented to the cellular system in a more native fashion.

Some embodiments of the present disclosure provide RNA (e.g., mRNA)vaccines that include at least one RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one antigenic polypeptideor an immunogenic fragment thereof (e.g., an immunogenic fragmentcapable of inducing an immune response to the antigenic polypeptide) andat least one RNA (e.g., mRNA polynucleotide) having an open readingframe encoding a flagellin adjuvant.

In some embodiments, at least one flagellin polypeptide (e.g., encodedflagellin polypeptide) is a flagellin protein. In some embodiments, atleast one flagellin polypeptide (e.g., encoded flagellin polypeptide) isan immunogenic flagellin fragment. In some embodiments, at least oneflagellin polypeptide and at least one antigenic polypeptide are encodedby a single RNA (e.g., mRNA) polynucleotide. In other embodiments, atleast one flagellin polypeptide and at least one antigenic polypeptideare each encoded by a different RNA polynucleotide.

In some embodiments at least one flagellin polypeptide has at least 80%,at least 85%, at least 90%, or at least 95% identity to a flagellinpolypeptide having a sequence identified by any one of SEQ ID NO: 54-56.

Provided herein, in some embodiments, is a ribonucleic acid (RNA) (e.g.,mRNA) vaccine, comprising at least one (e.g., at least 2, 3, 4 or 5) RNA(e.g., mRNA) polynucleotide having an open reading frame encoding atleast one (e.g., at least 2, 3, 4 or 5) hMPV, PIV, RSV, MeV, or aBetaCoV (e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH, HCoV-HKU1) antigenic polypeptide, or any combinationof two or more of the foregoing antigenic polypeptides. Herein, use ofthe term “antigenic polypeptide” encompasses immunogenic fragments ofthe antigenic polypeptide (an immunogenic fragment that is induces (oris capable of inducing) an immune response to hMPV, PIV, RSV, MeV, or aBetaCoV), unless otherwise stated.

Also provided herein, in some embodiments, is a RNA (e.g., mRNA) vaccinecomprising at least one (e.g., at least 2, 3, 4 or 5) RNA polynucleotidehaving an open reading frame encoding at least one (e.g., at least 2, 3,4 or 5) hMPV, PIV, RSV, MeV, and/or a BetaCoV (e.g., MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenicpolypeptide or an immunogenic fragment thereof, linked to a signalpeptide.

Further provided herein, in some embodiments, is a nucleic acid (e.g.,DNA) encoding at least one (e.g., at least 2, 3, 4 or 5) hMPV, PIV, RSV,MeV, and/or a BetaCoV (e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E,HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) RNA (e.g., mRNA) polynucleotide.

Further still, provided herein, in some embodiments, is a method ofinducing an immune response in a subject, the method comprisingadministering to the subject a vaccine comprising at least one (e.g., atleast 2, 3, 4 or 5) RNA (e.g., mRNA) polynucleotide having an openreading frame encoding at least one (e.g., at least 2, 3, 4 or 5) hMPV,PIV, RSV, MeV, and/or a BetaCoV (e.g., MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenicpolypeptide, or any combination of two or more of the foregoingantigenic polypeptides.

hMPV/PIV3/RSV

In some embodiments, a RNA (e.g., mRNA) vaccine comprises at least oneRNA (e.g., mRNA) polynucleotide having an open reading frame encoding atleast one hMPV, PIV3 or RSV antigenic polypeptide. In some embodiments,at least one antigenic polypeptide is a hMPV, PIV3 or RSV polyprotein.In some embodiments, at least one antigenic polypeptide is major surfaceglycoprotein G or an immunogenic fragment thereof. In some embodiments,at least one antigenic polypeptide is Fusion (F) glycoprotein (e.g.,Fusion glycoprotein F0, F1 or F2) or an immunogenic fragment thereof. Insome embodiments, at least one antigenic polypeptide is major surfaceglycoprotein G or an immunogenic fragment thereof and F glycoprotein oran immunogenic fragment thereof. In some embodiments, the antigenicpolypeptide is nucleoprotein (N) or an immunogenic fragment thereof,phosphoprotein (P) or an immunogenic fragment thereof, large polymeraseprotein (L) or an immunogenic fragment thereof, matrix protein (M) or animmunogenic fragment thereof, small hydrophobic protein (SH) or animmunogenic fragment thereof nonstructural protein 1 (NS1) or animmunogenic fragment thereof, or nonstructural protein 2 (NS2) and animmunogenic fragment thereof.

In some embodiments, at least one hMPV antigenic polypeptide comprisesan amino acid sequence identified by any one of SEQ ID NO: 5-8 (Table 3;see also amino acid sequences of Table 4). In some embodiments, theamino acid sequence of the hMPV antigenic polypeptide is, or is afragment of, or is a homolog or variant having at least 80% (e.g., 85%,90%, 95%, 98%, 99%) identity to, the amino acid sequence identified byany one of SEQ ID NO: 5-8 (Table 3; see also amino acid sequences ofTable 4).

In some embodiments, at least one hMPV antigenic polypeptide is encodedby a nucleic acid sequence identified by any one of SEQ ID NO: 1-4(Table 2).

In some embodiments, at least one hMPV RNA (e.g., mRNA) polynucleotideis encoded by a nucleic acid sequence, or a fragment of a nucleotidesequence, identified by any one of SEQ ID NO: 1-4 (Table 2). In someembodiments, at least one hMPV RNA (e.g., mRNA) polynucleotide comprisesa nucleic acid sequence, or a fragment of a nucleotide sequence,identified by any one of SEQ ID NO: 57-60 (Table 2).

In some embodiments, at least one antigenic polypeptide is obtained fromhMPV strain CAN98-75 (CAN75) or the hMPV strain CAN97-83 (CAN83).

In some embodiments, at least one PIV3 antigenic polypeptide compriseshemagglutinin-neuraminidase, Fusion (F) glycoprotein, matrix protein(M), nucleocapsid protein (N), viral replicase (L), non-structural Vprotein, or an immunogenic fragment thereof.

In some embodiments, at least one PIV3 antigenic polypeptide comprisesan amino acid sequence identified by any one of SEQ ID NO: 12-13 (Table6; see also amino acid sequences of Table 7). In some embodiments, theamino acid sequence of the PIV3 antigenic polypeptide is, or is afragment of, or is a homolog or variant having at least 80% (e.g., 85%,90%, 95%, 98%, 99%) identity to, the amino acid sequence identified byany one of SEQ ID NO: 12-13 (Table 6; see also amino acid sequences ofTable 7).

In some embodiments, at least one PIV3 antigenic polypeptide is encodedby a nucleic acid sequence identified by any one of SEQ ID NO: 9-12(Table 5; see also nucleic acid sequences of Table 7).

In some embodiments, at least one PIV3 RNA (e.g., mRNA) polynucleotideis encoded by a nucleic acid sequence, or a fragment of a nucleotidesequence, identified by any one of SEQ ID NO: 9-12 (Table 5; see alsonucleic acid sequences of Table 7). In some embodiments, at least onePIV3 RNA (e.g., mRNA) polynucleotide comprises a nucleic acid sequence,or a fragment of a nucleotide sequence, identified by any one of SEQ IDNO: 61-64 (Table 5).

In some embodiments, at least one antigenic polypeptide is obtained fromPIV3 strain HPIV3/Homo sapiens/PER/FLA4815/2008.

In some embodiments, at least one RSV antigenic polypeptide comprises atleast one antigenic polypeptide that comprises glycoprotein G,glycoprotein F, or an immunogenic fragment thereof. In some embodiments,at least one RSV antigenic polypeptide comprises at least one antigenicpolypeptide that comprises glycoprotein F and at least one or at leasttwo antigenic polypeptide selected from G, M, N, P, L, SH, M2, NS1 andNS2.

MeV

In some embodiments, a RNA (e.g., mRNA) vaccine comprises at least oneRNA (e.g., mRNA) polynucleotide having an open reading frame encoding atleast one MeV antigenic polypeptide. In some embodiments, at least oneantigenic polypeptide is a hemagglutinin (HA) protein or an immunogenicfragment thereof. The HA protein may be from MeV strain D3 or B8, forexample. In some embodiments, at least one antigenic polypeptide is aFusion (F) protein or an immunogenic fragment thereof. The F protein maybe from MeV strain D3 or B8, for example. In some embodiments, a MeV RNA(e.g., mRNA) vaccines comprises a least one RNA polynucleotide encodinga HA protein and a F protein. The HA and F proteins may be from MeVstrain D3 or B8, for example.

In some embodiments, at least one MeV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 47-50 (Table14). In some embodiments, the amino acid sequence of the MeV antigenicpolypeptide is, or is a fragment of, or is a homolog or variant havingat least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to, the amino acidsequence identified by any one of SEQ ID NO: 47-50 (Table 14).

In some embodiments, at least one MeV antigenic polypeptide is encodedby a nucleic acid sequence of SEQ ID NO: 35-46 (Table 13).

In some embodiments, at least one MeV RNA (e.g., mRNA) polynucleotide isencoded by a nucleic acid sequence, or a fragment of a nucleotidesequence, identified by any one of SEQ ID NO: 35-46 (Table 13). In someembodiments, at least one MeV RNA (e.g., mRNA) polynucleotide comprisesa nucleic acid sequence, or a fragment of a nucleotide sequence,identified by any one of SEQ ID NO: 69-80 (Table 13).

In some embodiments, at least one antigenic polypeptide is obtained fromMeV strain B3/B3.1, C2, D4, D6, D7, D8, G3, H1, Moraten, Rubeovax,MVi/New Jersey.USA/45.05, MVi/Texas.USA/4.07, AIK-C, MVi/NewYork.USA/26.09/3, MVi/California.USA/16.03, MVi/Virginia.USA/15.09,MVi/California.USA/8.04, or MVi/Pennsylvania.USA/20.09.

BetaCoV

In some embodiments, a RNA (e.g., mRNA) vaccine comprises at least oneRNA (e.g., mRNA) polynucleotide having an open reading frame encoding atleast one BetaCoV antigenic polypeptide. In some embodiments, theBetaCoV is MERS-CoV. In some embodiments, the BetaCoV is SARS-CoV. Insome embodiments, the BetaCoV is HCoV-OC43. In some embodiments, theBetaCoV is HCoV-229E. In some embodiments, the BetaCoV is HCoV-NL63. Insome embodiments, the BetaCoV is HCoV-HKU1. In some embodiments, atleast one antigenic polypeptide is a Betacoronavirus structural protein.For example, a Betacoronavirus structural protein may be spike protein(S), envelope protein (E), nucleocapsid protein (N), membrane protein(M) or an immunogenic fragment thereof. In some embodiments, aBetacoronavirus structural protein is a spike protein (S). In someembodiments, a Betacoronavirus structural protein is a S1 subunit or aS2 subunit of spike protein (S) or an immunogenic fragment thereof.

BetaCoV RNA (e.g., mRNA) polynucleotides of the vaccines provided hereinmay encode viral protein components of Betacoronaviruses, for example,accessory proteins, replicase proteins and the like are encompassed bythe present disclosure. RNA (e.g., mRNA) vaccines may include RNApolynucleotides encoding at least one accessory protein (e.g., protein3, protein 4a, protein 4b, protein 5), at least one replicase protein(e.g., protein 1a, protein 1b), or a combination of at least oneaccessory protein and at least one replicase protein. The presentdisclosure also encompasses RNA (e.g., mRNA) vaccines comprising RNA(e.g., mRNA) polynucleotides encoding an accessory protein and/or areplicase protein in combination with at least one structural protein.Due to their surface expression properties, vaccines featuring RNApolynucleotides encoding structural proteins are believed to havepreferred immunogenic activity and, hence, may be most suitable for usein the vaccines of the present disclosure.

Some embodiments of the present disclosure provide Betacoronavirus(e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH, HCoV-HKU1 or a combination thereof) vaccines that include atleast one RNA (e.g., mRNA) polynucleotide having an open reading frameencoding at least one Betacoronavirus (e.g., MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1) antigenicpolypeptide. Also provided herein are pan-Betacoronavirus vaccines.Thus, a Betacoronavirus vaccine comprising a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding any one, two, threeor four of MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, andHCoV-HKU1, for example, may be effective against any one of, anycombination of, or all of, MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E,HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1. Other Betacoronaviruses areencompassed by the present disclosure.

In some embodiments, at least one antigenic polypeptide is a MERS-CoVstructural protein. For example, a MERS-CoV structural protein may bespike protein (S), envelope protein (E), nucleocapsid protein (N),membrane protein (M) or an immunogenic fragment thereof. In someembodiments, the MERS-CoV structural protein is a spike protein (S)(see, e.g., Coleman C M et al. Vaccine 2014; 32:3169-74, incorporatedherein by reference). In some embodiments, the MERS-CoV structuralprotein is a S1 subunit or a S2 subunit of spike protein (S) or animmunogenic fragment thereof (Li J et al. Viral Immunol 2013;26(2):126-32; He Y et al. Biochem Biophys Res Commun 2004;324(2):773-81, each of which is incorporated herein by reference).

In some embodiments, at least one MERS-CoV antigenic polypeptidecomprises an amino acid sequence identified by any one of SEQ ID NO:24-28 or 33 (Table 11). In some embodiments, the amino acid sequence ofthe MERS-CoV antigenic polypeptide is, or is a fragment of, or is ahomolog or variant having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%)identity to, the amino acid sequence identified by any one of SEQ ID NO:24-28 or 33 (Table 11).

In some embodiments, at least one MERS-CoV antigenic polypeptide isencoded by a nucleic acid sequence identified by any one of SEQ ID NO:20-23 (Table 10).

In some embodiments, at least one MERS-CoV RNA (e.g., mRNA)polynucleotide is encoded by a nucleic acid sequence, or a fragment of anucleotide sequence, identified by any one of SEQ ID NO: 20-23 (Table10). In some embodiments, at least one MERS-CoV RNA (e.g., mRNA)polynucleotide comprises a nucleic acid sequence, or a fragment of anucleotide sequence, identified by any one of SEQ ID NO: 65-68 (Table10).

In some embodiments, at least one antigenic polypeptide is obtained fromMERS-CoV strain Riyadh_14_2013, 2cEMC/2012, or Hasa_1_2013.

In some embodiments, at least one antigenic polypeptide is a SARS-CoVstructural protein. For example, a SARS-CoV structural protein may bespike protein (S), envelope protein (E), nucleocapsid protein (N),membrane protein (M) or an immunogenic fragment thereof. In someembodiments, the SARS-CoV structural protein is a spike protein (S). Insome embodiments, the SARS-CoV structural protein is a S1 subunit or aS2 subunit of spike protein (S) or an immunogenic fragment thereof.

In some embodiments, at least one SARS-CoV antigenic polypeptidecomprises an amino acid sequence identified by any one of SEQ ID NO: 29,32 or 34 (Table 11). In some embodiments, the amino acid sequence of theSARS-CoV antigenic polypeptide is, or is a fragment of, or is a homologor variant having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identityto, the amino acid sequence identified by any one of SEQ ID NO: 29, 32or 34 (Table 11).

In some embodiments, at least one antigenic polypeptide is a HCoV-OC43structural protein. For example, a HCoV-OC43 structural protein may bespike protein (S), envelope protein (E), nucleocapsid protein (N),membrane protein (M) or an immunogenic fragment thereof. In someembodiments, the HCoV-OC43 structural protein is a spike protein (S). Insome embodiments, the HCoV-OC43 structural protein is a S1 subunit or aS2 subunit of spike protein (S) or an immunogenic fragment thereof.

In some embodiments, at least one HCoV-OC43 antigenic polypeptidecomprises an amino acid sequence identified by any one of SEQ ID NO: 30(Table 11). In some embodiments, the amino acid sequence of theHCoV-OC43 antigenic polypeptide is, or is a fragment of, or is a homologor variant having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identityto, the amino acid sequence identified by any one of SEQ ID NO: 30(Table 11).

In some embodiments, an antigenic polypeptide is a HCoV-HKU1 structuralprotein. For example, a HCoV-HKU1 structural protein may be spikeprotein (S), envelope protein (E), nucleocapsid protein (N), membraneprotein (M) or an immunogenic fragment thereof. In some embodiments, theHCoV-HKU1 structural protein is a spike protein (S). In someembodiments, the HCoV-HKU1 structural protein is a S1 subunit or a S2subunit of spike protein (S) or an immunogenic fragment thereof.

In some embodiments, at least one HCoV-HKU1 antigenic polypeptidecomprises an amino acid sequence identified by any one of SEQ ID NO: 31(Table 11). In some embodiments, the amino acid sequence of theHCoV-HKU1 antigenic polypeptide is, or is a fragment of, or is a homologor variant having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identityto, the amino acid sequence identified by any one of SEQ ID NO: 31(Table 11).

In some embodiments, an open reading frame of a RNA (e.g., mRNA) vaccineis codon-optimized. In some embodiments, at least one RNA polynucleotideencodes at least one antigenic polypeptide having an amino acid sequenceidentified by any one of SEQ ID NO: 5-8, 12-13, 24-34, or 47-50 (Tables3, 6, 11 and 14; see also amino acid sequences of Tables 4, 7, 12 and15) and is codon optimized mRNA.

In some embodiments, a RNA (e.g., mRNA) vaccine further comprising anadjuvant.

Tables 4, 7, 12 and 15 provide National Center for BiotechnologyInformation (NCBI) accession numbers of interest. It should beunderstood that the phrase “an amino acid sequence of Tables 4, 7, 12and 15” refers to an amino acid sequence identified by one or more NCBIaccession numbers listed in Tables 4, 7, 12 and 15. Each of the aminoacid sequences, and variants having greater than 95% identity or greaterthan 98% identity to each of the amino acid sequences encompassed by theaccession numbers of Tables 4, 7, 12 and 15 are included within theconstructs (polynucleotides/polypeptides) of the present disclosure.

In some embodiments, at least one mRNA polynucleotide is encoded by anucleic acid having a sequence identified by any one of SEQ ID NO: 1-4,9-12, 20-23, or 35-46 (Tables 2, 5, 10 and 13; see also nucleic acidsequences of Table 7) and having less than 80% identity to wild-typemRNA sequence. In some embodiments, at least one mRNA polynucleotide isencoded by a nucleic acid having a sequence identified by any one of SEQID NO: 1-4, 9-12, 20-23, or 35-46 (Tables 2, 5, 10 and 13; see alsonucleic acid sequences of Table 7) and having less than 75%, 85% or 95%identity to a wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide is encoded by a nucleic acid having a sequenceidentified by any one of SEQ ID NO: 1-4, 9-12, 20-23, or 35-46 (Tables2, 5, 10 and 13; see also nucleic acid sequences of Table 7) and havingless than 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80%identity to wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide is encoded by a nucleic acid having a sequenceidentified by any one of SEQ ID NO: 1-4, 9-12, 20-23, or 35-46 (Tables2, 5, 10 and 13; see also nucleic acid sequences of Table 7) and havingless than 40-85%, 50-85%, 60-85%, 30-85%, 70-85%, 75-85% or 80-85%identity to wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide is encoded by a nucleic acid having a sequenceidentified by any one of SEQ ID NO: 1-4, 9-12, 20-23, or 35-46 (Tables2, 5, 10 and 13; see also nucleic acid sequences of Table 7) and havingless than 40-90%, 50-90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or85-90% identity to wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having an amino acid sequence identified byany one of SEQ ID NO: 5-8, 12-13, 24-34, or 47-50 (Tables 3, 6, 11 and14; see also amino acid sequences of Tables 4, 7, 12 and 15) and havingat least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to wild-type mRNAsequence, but does not include wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having an amino acid sequence identified byany one of SEQ ID NO: 5-8, 12-13, 24-34, or 47-50 (Tables 3, 6, 11 and14; see also amino acid sequences of Tables 4, 7, 12 and 15) and hasless than 95%, 90%, 85%, 80% or 75% identity to wild-type mRNA sequence.In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having an amino acid sequence identified byany one of SEQ ID NO: 5-8, 12-13, 24-34, or 47-50 (Tables 3, 6, 11 and14; see also amino acid sequences of Tables 4, 7, 12 and 15) and has30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 75-80% or 78-80%, 30-85%,40-85%, 50-805%, 60-85%, 70-85%, 75-85% or 78-85%, 30-90%, 40-90%,50-90%, 60-90%, 70-90%, 75-90%, 80-90% or 85-90% identity to wild-typemRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to an aminoacid sequence identified by any one of SEQ ID NO: 5-8, 12-13, 24-34, or47-50 (Tables 3, 6, 11 and 14; see also amino acid sequences of Tables4, 7, 12 and 15). In some embodiments, at least one RNA polynucleotideencodes at least one antigenic polypeptide having 95%-99% identity to anamino acid sequence identified by any one of SEQ ID NO: 5-8, 12-13,24-34, or 47-50 (Tables 3, 6, 11 and 14; see also amino acid sequencesof Tables 4, 7, 12 and 15).

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide having at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to an aminoacid sequence identified by any one of SEQ ID NO: 5-8, 12-13, 24-34, or47-50 (Tables 3, 6, 11 and 14; see also amino acid sequences of Tables4, 7, 12 and 15) and having membrane fusion activity. In someembodiments, at least one RNA polynucleotide encodes at least oneantigenic polypeptide having 95%-99% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 5-8, 12-13, 24-34, or 47-50 (Tables3, 6, 11 and 14; see also amino acid sequences of Tables 4, 7, 12 and15) and having membrane fusion activity.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one hMPV antigenicpolypeptide, at least one PIV3 antigenic polypeptide, at least one RSVantigenic polypeptide, at least one MeV antigenic polypeptide, or atleast one BetaCoV antigenic polypeptide, e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1, or any combination of two or more of the foregoing antigenicpolypeptides) that attaches to cell receptors.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one hMPV antigenicpolypeptide, at least one PIV3 antigenic polypeptide, at least one RSVantigenic polypeptide, at least one MeV antigenic polypeptide, or atleast one BetaCoV antigenic polypeptide, e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1, or any combination of two or more of the foregoing antigenicpolypeptides) that causes fusion of viral and cellular membranes.

In some embodiments, at least one RNA polynucleotide encodes at leastone antigenic polypeptide (e.g., at least one hMPV antigenicpolypeptide, at least one PIV3 antigenic polypeptide, at least one RSVantigenic polypeptide, at least one MeV antigenic polypeptide, or atleast one BetaCoV antigenic polypeptide, e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1, or any combination of two or more of the foregoing antigenicpolypeptides) that is responsible for binding of the virus to a cellbeing infected.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one antigenic polypeptide(e.g., at least one hMPV antigenic polypeptide, at least one PIV3antigenic polypeptide, at least one RSV antigenic polypeptide, at leastone MeV antigenic polypeptide, or at least one BetaCoV antigenicpolypeptide, e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1, or any combinationof two or more of the foregoing antigenic polypeptides), at least one 5′terminal cap and at least one chemical modification, formulated within alipid nanoparticle.

In some embodiments, a 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.

In some embodiments, at least one chemical modification is selected frompseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, the chemical modification is in the 5-position of theuracil. In some embodiments, the chemical modification is aN1-methylpseudouridine. In some embodiments, the chemical modificationis a N1-ethylpseudouridine.

In some embodiments, a lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid. In someembodiments, a cationic lipid is an ionizable cationic lipid and thenon-cationic lipid is a neutral lipid, and the sterol is a cholesterol.In some embodiments, a cationic lipid is selected from the groupconsisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319),(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), andN,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

In some embodiments, the lipid is

In some embodiments, the lipid is

In some embodiments, a lipid nanoparticle comprises compounds of Formula(I) and/or Formula (II), discussed below.

In some embodiments, a respiratory virus RNA (e.g., mRNA) vaccine isformulated in a lipid nanoparticle that comprises a compound selectedfrom Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 and 122, describedbelow.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA (e.g., mRNA) polynucleotide having an openreading frame encoding at least one antigenic polypeptide (e.g., atleast one hMPV antigenic polypeptide, at least one PIV3 antigenicpolypeptide, at least one RSV antigenic polypeptide, at least one MeVantigenic polypeptide, or at least one BetaCoV antigenic polypeptide,e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1, or any combination of two or more of theforegoing antigenic polypeptides), wherein at least 80% (e.g., 85%, 90%,95%, 98%, 99%) of the uracil in the open reading frame have a chemicalmodification, optionally wherein the vaccine is formulated in a lipidnanoparticle (e.g., a lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid).

In some embodiments, 100% of the uracil in the open reading frame have achemical modification. In some embodiments, a chemical modification isin the 5-position of the uracil. In some embodiments, a chemicalmodification is a N1-methyl pseudouridine. In some embodiments, 100% ofthe uracil in the open reading frame have a N1-methyl pseudouridine inthe 5-position of the uracil.

In some embodiments, an open reading frame of a RNA (e.g., mRNA)polynucleotide encodes at least two antigenic polypeptides (e.g., atleast two hMPV antigenic polypeptides, at least two PIV3 antigenicpolypeptides, at least two RSV antigenic polypeptides, at least two MeVantigenic polypeptides, or at least two BetaCoV antigenic polypeptides,e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1, or any combination of two or more of theforegoing antigenic polypeptides). In some embodiments, the open readingframe encodes at least five or at least ten antigenic polypeptides. Insome embodiments, the open reading frame encodes at least 100 antigenicpolypeptides. In some embodiments, the open reading frame encodes 2-100antigenic polypeptides.

In some embodiments, a vaccine comprises at least two RNA (e.g., mRNA)polynucleotides, each having an open reading frame encoding at least oneantigenic polypeptide (e.g., at least one hMPV antigenic polypeptide, atleast one PIV3 antigenic polypeptide, at least one RSV antigenicpolypeptide, at least one MeV antigenic polypeptide, or at least oneBetaCoV antigenic polypeptide, e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1, or anycombination of two or more of the foregoing antigenic polypeptides). Insome embodiments, the vaccine comprises at least five or at least tenRNA (e.g., mRNA) polynucleotides, each having an open reading frameencoding at least one antigenic polypeptide or an immunogenic fragmentthereof. In some embodiments, the vaccine comprises at least 100 RNA(e.g., mRNA) polynucleotides, each having an open reading frame encodingat least one antigenic polypeptide. In some embodiments, the vaccinecomprises 2-100 RNA (e.g., mRNA) polynucleotides, each having an openreading frame encoding at least one antigenic polypeptide.

In some embodiments, at least one antigenic polypeptide (e.g., at leastone hMPV antigenic polypeptide, at least one PIV3 antigenic polypeptide,at least one RSV antigenic polypeptide, at least one MeV antigenicpolypeptide, or at least one BetaCoV antigenic polypeptide, e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1, or any combination of two or more of theforegoing antigenic polypeptides) is fused to a signal peptide. In someembodiments, the signal peptide is selected from: a HuIgGk signalpeptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO: 15); IgE heavy chain epsilon-1signal peptide (MDWTWILFLVAAATRVHS; SEQ ID NO: 16); Japaneseencephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO:17), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 18) andJapanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO:19).

In some embodiments, the signal peptide is fused to the N-terminus of atleast one antigenic polypeptide. In some embodiments, a signal peptideis fused to the C-terminus of at least one antigenic polypeptide.

In some embodiments, at least one antigenic polypeptide (e.g., at leastone hMPV antigenic polypeptide, at least one PIV3 antigenic polypeptide,at least one RSV antigenic polypeptide, at least one MeV antigenicpolypeptide, or at least one BetaCoV antigenic polypeptide, e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1, or any combination of two or more of theforegoing antigenic polypeptides) comprises a mutated N-linkedglycosylation site.

Also provided herein is a RNA (e.g., mRNA) vaccine of any one of theforegoing paragraphs (e.g., a hMPV vaccine, a PIV3 vaccine, a RSVvaccine, a MeV vaccine, or a BetaCoV vaccine, e.g., selected fromMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand HCoV-HKU1, or any combination of two or more of the foregoingvaccines), formulated in a nanoparticle (e.g., a lipid nanoparticle).

In some embodiments, the nanoparticle has a mean diameter of 50-200 nm.In some embodiments, the nanoparticle is a lipid nanoparticle. In someembodiments, the lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid. In someembodiments, the lipid nanoparticle comprises a molar ratio of about20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and25% non-cationic lipid. In some embodiments, the cationic lipid is anionizable cationic lipid and the non-cationic lipid is a neutral lipid,and the sterol is a cholesterol. In some embodiments, the cationic lipidis selected from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

In some embodiments, a lipid nanoparticle comprises compounds of Formula(I) and/or Formula (II), as discussed below.

In some embodiments, a lipid nanoparticle comprises Compounds 3, 18, 20,25, 26, 29, 30, 60, 108-112, or 122, as discussed below.

In some embodiments, the nanoparticle has a polydispersity value of lessthan 0.4 (e.g., less than 0.3, 0.2 or 0.1).

In some embodiments, the nanoparticle has a net neutral charge at aneutral pH value.

In some embodiments, the respiratory virus vaccine is multivalent.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, comprisingadministering to the subject any of the RNA (e.g., mRNA) vaccine asprovided herein in an amount effective to produce an antigen-specificimmune response. In some embodiments, the RNA (e.g., mRNA) vaccine is ahMPV vaccine, a PIV3 vaccine, a RSV vaccine, a MeV vaccine, or a BetaCoVvaccine, e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E,HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1 vaccines. In some embodiments,the RNA (e.g., mRNA) vaccine is a combination vaccine comprising acombination of any two or more of the foregoing vaccines.

In some embodiments, an antigen-specific immune response comprises a Tcell response or a B cell response.

In some embodiments, a method of producing an antigen-specific immuneresponse comprises administering to a subject a single dose (no boosterdose) of a RNA (e.g., mRNA) vaccine of the present disclosure. In someembodiments, the RNA (e.g., mRNA) vaccine is a hMPV vaccine, a PIV3vaccine, a RSV vaccine, a MeV vaccine, or a BetaCoV vaccine, e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1 vaccines. In some embodiments, the RNA(e.g., mRNA) vaccine is a combination vaccine comprising a combinationof any two or more of the foregoing vaccines.

In some embodiments, a method further comprises administering to thesubject a second (booster) dose of a RNA (e.g., mRNA) vaccine.Additional doses of a RNA (e.g., mRNA) vaccine may be administered.

In some embodiments, the subjects exhibit a seroconversion rate of atleast 80% (e.g., at least 85%, at least 90%, or at least 95%) followingthe first dose or the second (booster) dose of the vaccine.Seroconversion is the time period during which a specific antibodydevelops and becomes detectable in the blood. After seroconversion hasoccurred, a virus can be detected in blood tests for the antibody.During an infection or immunization, antigens enter the blood, and theimmune system begins to produce antibodies in response. Beforeseroconversion, the antigen itself may or may not be detectable, butantibodies are considered absent. During seroconversion, antibodies arepresent but not yet detectable. Any time after seroconversion, theantibodies can be detected in the blood, indicating a prior or currentinfection.

In some embodiments, a RNA (e.g., mRNA) vaccine is administered to asubject by intradermal or intramuscular injection.

Some embodiments, of the present disclosure provide methods of inducingan antigen specific immune response in a subject, includingadministering to a subject a RNA (e.g., mRNA) vaccine in an effectiveamount to produce an antigen specific immune response in a subject.Antigen-specific immune responses in a subject may be determined, insome embodiments, by assaying for antibody titer (for titer of anantibody that binds to a hMPV, PIV3, RSV, MeV and/or BetaCoV antigenicpolypeptide) following administration to the subject of any of the RNA(e.g., mRNA) vaccines of the present disclosure. In some embodiments,the anti-antigenic polypeptide antibody titer produced in the subject isincreased by at least 1 log relative to a control. In some embodiments,the anti-antigenic polypeptide antibody titer produced in the subject isincreased by 1-3 log relative to a control.

In some embodiments, the anti-antigenic polypeptide antibody titerproduced in a subject is increased at least 2 times relative to acontrol. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased at least 5 times relative toa control. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased at least 10 times relative toa control. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased 2-10 times relative to acontrol.

In some embodiments, the control is an anti-antigenic polypeptideantibody titer produced in a subject who has not been administered a RNA(e.g., mRNA) vaccine of the present disclosure. In some embodiments, thecontrol is an anti-antigenic polypeptide antibody titer produced in asubject who has been administered a live attenuated or inactivated hMPV,PIV3, RSV, MeV and/or BetaCoV vaccine (see, e.g., Ren J. et al. J ofGen. Virol. 2015; 96: 1515-1520), or wherein the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasbeen administered a recombinant or purified hMPV, PIV3, RSV, MeV and/orBetaCoV protein vaccine. In some embodiments, the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasbeen administered a hMPV, PIV3, RSV, MeV and/or BetaCoV virus-likeparticle (VLP) vaccine (see, e.g., Cox R G et al., J Virol. 2014 June;88(11): 6368-6379).

A RNA (e.g., mRNA) vaccine of the present disclosure is administered toa subject in an effective amount (an amount effective to induce animmune response). In some embodiments, the effective amount is a doseequivalent to an at least 2-fold, at least 4-fold, at least 10-fold, atleast 100-fold, at least 1000-fold reduction in the standard of caredose of a recombinant hMPV, PIV3, RSV, MeV and/or BetaCoV proteinvaccine, wherein the anti-antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a recombinant hMPV, PIV3, RSV, MeV and/or BetaCoV proteinvaccine, a purified hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine,a live attenuated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine, aninactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine, or a hMPV,PIV3, RSV, MeV and/or BetaCoV VLP vaccine. In some embodiments, theeffective amount is a dose equivalent to 2-1000-fold reduction in thestandard of care dose of a recombinant hMPV, PIV3, RSV, MeV and/orBetaCoV protein vaccine, wherein the anti-antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant hMPV, PIV3, RSV, MeV and/orBetaCoV protein vaccine, a purified hMPV, PIV3, RSV, MeV and/or BetaCoVprotein vaccine, a live attenuated hMPV, PIV3, RSV, MeV and/or BetaCoVvaccine, an inactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine, ora hMPV, PIV3, RSV, MeV and/or BetaCoV VLP vaccine.

In some embodiments, the control is an anti-antigenic polypeptideantibody titer produced in a subject who has been administered avirus-like particle (VLP) vaccine comprising structural proteins ofhMPV, PIV3, RSV, MeV and/or BetaCoV.

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated in aneffective amount to produce an antigen specific immune response in asubject.

In some embodiments, the effective amount is a total dose of 25 μg to1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amountis a total dose of 100 μg. In some embodiments, the effective amount isa dose of 25 μg administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 100 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 400 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 500 μgadministered to the subject a total of two times.

In some embodiments, the efficacy (or effectiveness) of a RNA (e.g.,mRNA) vaccine is greater than 60%. In some embodiments, the RNA (e.g.,mRNA) polynucleotide of the vaccine at least one hMPV antigenicpolypeptide, at least one PIV3 antigenic polypeptide, at least one RSVantigenic polypeptide, at least one MeV antigenic polypeptide, at leastone BetaCoV antigenic polypeptide, e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1, or any combination of two or more of the foregoing antigenicpolypeptides.

Vaccine efficacy may be assessed using standard analyses (see, e.g.,Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). Forexample, vaccine efficacy may be measured by double-blind, randomized,clinical controlled trials. Vaccine efficacy may be expressed as aproportionate reduction in disease attack rate (AR) between theunvaccinated (ARU) and vaccinated (ARV) study cohorts and can becalculated from the relative risk (RR) of disease among the vaccinatedgroup with use of the following formulas:Efficacy=(ARU−ARV)/ARU×100; andEfficacy=(1−RR)×100.

Likewise, vaccine effectiveness may be assessed using standard analyses(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;201(11):1607-10). Vaccine effectiveness is an assessment of how avaccine (which may have already proven to have high vaccine efficacy)reduces disease in a population. This measure can assess the net balanceof benefits and adverse effects of a vaccination program, not just thevaccine itself, under natural field conditions rather than in acontrolled clinical trial. Vaccine effectiveness is proportional tovaccine efficacy (potency) but is also affected by how well targetgroups in the population are immunized, as well as by othernon-vaccine-related factors that influence the ‘real-world’ outcomes ofhospitalizations, ambulatory visits, or costs. For example, aretrospective case control analysis may be used, in which the rates ofvaccination among a set of infected cases and appropriate controls arecompared. Vaccine effectiveness may be expressed as a rate difference,with use of the odds ratio (OR) for developing infection despitevaccination:Effectiveness=(1−OR)×100.

In some embodiments, the efficacy (or effectiveness) of a RNA (e.g.,mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, or at least 90%.

In some embodiments, the vaccine immunizes the subject against hMPV,PIV3, RSV, MeV, BetaCoV (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1), or anycombination of two or more of the foregoing viruses for up to 2 years.In some embodiments, the vaccine immunizes the subject against hMPV,PIV3, RSV, MeV, BetaCoV (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1), or anycombination of two or more of the foregoing viruses for more than 2years, more than 3 years, more than 4 years, or for 5-10 years.

In some embodiments, the subject is about 5 years old or younger. Forexample, the subject may be between the ages of about 1 year and about 5years (e.g., about 1, 2, 3, 5 or 5 years), or between the ages of about6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months).In some embodiments, the subject is about 12 months or younger (e.g.,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In someembodiments, the subject is about 6 months or younger.

In some embodiments, the subject was born full term (e.g., about 37-42weeks). In some embodiments, the subject was born prematurely, forexample, at about 36 weeks of gestation or earlier (e.g., about 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 weeks). For example, thesubject may have been born at about 32 weeks of gestation or earlier. Insome embodiments, the subject was born prematurely between about 32weeks and about 36 weeks of gestation. In such subjects, a RNA (e.g.,mRNA) vaccine may be administered later in life, for example, at the ageof about 6 months to about 5 years, or older.

In some embodiments, the subject is pregnant (e.g., in the first, secondor third trimester) when administered an RNA (e.g., mRNA) vaccine.Viruses such as hMPV, PIV3 and RSV causes infections of the lowerrespiratory tract, mainly in infants and young children. One-third ofRSV related deaths, for example, occur in the first year of life, with99 percent of these deaths occurring in low-resource countries. It's sowidespread in the United States that nearly all children become infectedwith the virus before their second birthdays. Thus, the presentdisclosure provides RNA (e.g., mRNA) vaccines for maternal immunizationto improve mother-to-child transmission of protection against the virus.

In some embodiments, the subject is a young adult between the ages ofabout 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or50 years old).

In some embodiments, the subject is an elderly subject about 60 yearsold, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85 or90 years old).

In some embodiments, the subject is has a chronic pulmonary disease(e.g., chronic obstructive pulmonary disease (COPD) or asthma). Twoforms of COPD include chronic bronchitis, which involves a long-termcough with mucus, and emphysema, which involves damage to the lungs overtime. Thus, a subject administered a RNA (e.g., mRNA) vaccine may havechronic bronchitis or emphysema.

In some embodiments, the subject has been exposed to hMPV, PIV3, RSV,MeV, BetaCoV (e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1), or anycombination of two or more of the foregoing viruses; the subject isinfected with hMPV, PIV3, RSV, MeV, BetaCoV (e.g., selected fromMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand HCoV-HKU1), or any combination of two or more of the foregoingviruses; or subject is at risk of infection by hMPV, PIV3, RSV, MeV,BetaCoV (e.g., selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E,HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1), or any combination of two ormore of the foregoing viruses.

In some embodiments, the subject is immunocompromised (has an impairedimmune system, e.g., has an immune disorder or autoimmune disorder).

In some embodiments the nucleic acid vaccines described herein arechemically modified. In other embodiments the nucleic acid vaccines areunmodified.

Yet other aspects provide compositions for and methods of vaccinating asubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a first respiratory virus antigenic polypeptide, wherein theRNA polynucleotide does not include a stabilization element, and whereinan adjuvant is not coformulated or co-administered with the vaccine.

In other aspects the invention is a composition for or method ofvaccinating a subject comprising administering to the subject a nucleicacid vaccine comprising one or more RNA polynucleotides having an openreading frame encoding a first antigenic polypeptide wherein a dosage ofbetween 10 μg/kg and 400 μg/kg of the nucleic acid vaccine isadministered to the subject. In some embodiments the dosage of the RNApolynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg,100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg,80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg,300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or300-400 μg per dose. In some embodiments, the nucleic acid vaccine isadministered to the subject by intradermal or intramuscular injection.In some embodiments, the nucleic acid vaccine is administered to thesubject on day zero. In some embodiments, a second dose of the nucleicacid vaccine is administered to the subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 100 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 50 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 75 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 150 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 400 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 200 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, the RNA polynucleotide accumulates at a 100 foldhigher level in the local lymph node in comparison with the distal lymphnode. In other embodiments the nucleic acid vaccine is chemicallymodified and in other embodiments the nucleic acid vaccine is notchemically modified.

Aspects of the invention provide a nucleic acid vaccine comprising oneor more RNA polynucleotides having an open reading frame encoding afirst antigenic polypeptide, wherein the RNA polynucleotide does notinclude a stabilization element, and a pharmaceutically acceptablecarrier or excipient, wherein an adjuvant is not included in thevaccine. In some embodiments, the stabilization element is a histonestem-loop. In some embodiments, the stabilization element is a nucleicacid sequence having increased GC content relative to wild typesequence.

Aspects of the invention provide nucleic acid vaccines comprising one ormore RNA polynucleotides having an open reading frame encoding a firstantigenic polypeptide, wherein the RNA polynucleotide is present in theformulation for in vivo administration to a host, which confers anantibody titer superior to the criterion for seroprotection for thefirst antigen for an acceptable percentage of human subjects. In someembodiments, the antibody titer produced by the mRNA vaccines of theinvention is a neutralizing antibody titer. In some embodiments theneutralizing antibody titer is greater than a protein vaccine. In otherembodiments the neutralizing antibody titer produced by the mRNAvaccines of the invention is greater than an adjuvanted protein vaccine.In yet other embodiments the neutralizing antibody titer produced by themRNA vaccines of the invention is 1,000-10,000, 1,200-10,000,1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000,2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000,3,000-4,000, or 2,000-2,500. A neutralization titer is typicallyexpressed as the highest serum dilution required to achieve a 50%reduction in the number of plaques.

Also provided are nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide, wherein the RNA polynucleotide is present in a formulationfor in vivo administration to a host for eliciting a longer lasting highantibody titer than an antibody titer elicited by an mRNA vaccine havinga stabilizing element or formulated with an adjuvant and encoding thefirst antigenic polypeptide. In some embodiments, the RNA polynucleotideis formulated to produce a neutralizing antibodies within one week of asingle administration. In some embodiments, the adjuvant is selectedfrom a cationic peptide and an immunostimulatory nucleic acid. In someembodiments, the cationic peptide is protamine.

Aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no nucleotide modification, the openreading frame encoding a first antigenic polypeptide, wherein the RNApolynucleotide is present in the formulation for in vivo administrationto a host such that the level of antigen expression in the hostsignificantly exceeds a level of antigen expression produced by an mRNAvaccine having a stabilizing element or formulated with an adjuvant andencoding the first antigenic polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no nucleotide modification, the openreading frame encoding a first antigenic polypeptide, wherein thevaccine has at least 10 fold less RNA polynucleotide than is requiredfor an unmodified mRNA vaccine to produce an equivalent antibody titer.In some embodiments, the RNA polynucleotide is present in a dosage of25-100 micrograms.

Aspects of the invention also provide a unit of use vaccine, comprisingbetween 10 ug and 400 ug of one or more RNA polynucleotides having anopen reading frame comprising at least one chemical modification oroptionally no nucleotide modification, the open reading frame encoding afirst antigenic polypeptide, and a pharmaceutically acceptable carrieror excipient, formulated for delivery to a human subject. In someembodiments, the vaccine further comprises a cationic lipidnanoparticle.

Aspects of the invention provide methods of creating, maintaining orrestoring antigenic memory to a respiratory virus strain in anindividual or population of individuals comprising administering to saidindividual or population an antigenic memory booster nucleic acidvaccine comprising (a) at least one RNA polynucleotide, saidpolynucleotide comprising at least one chemical modification oroptionally no nucleotide modification and two or more codon-optimizedopen reading frames, said open reading frames encoding a set ofreference antigenic polypeptides, and (b) optionally a pharmaceuticallyacceptable carrier or excipient. In some embodiments, the vaccine isadministered to the individual via a route selected from the groupconsisting of intramuscular administration, intradermal administrationand subcutaneous administration. In some embodiments, the administeringstep comprises contacting a muscle tissue of the subject with a devicesuitable for injection of the composition. In some embodiments, theadministering step comprises contacting a muscle tissue of the subjectwith a device suitable for injection of the composition in combinationwith electroporation.

Aspects of the invention provide methods of vaccinating a subjectcomprising administering to the subject a single dosage of between 25ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide in an effective amount to vaccinate the subject.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification, the open reading frame encoding a first antigenicpolypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine toproduce an equivalent antibody titer. In some embodiments, the RNApolynucleotide is present in a dosage of 25-100 micrograms.

Other aspects provide nucleic acid vaccines comprising an LNP formulatedRNA polynucleotide having an open reading frame comprising no nucleotidemodifications (unmodified), the open reading frame encoding a firstantigenic polypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine notformulated in a LNP to produce an equivalent antibody titer. In someembodiments, the RNA polynucleotide is present in a dosage of 25-100micrograms.

The data presented in the Examples demonstrate significant enhancedimmune responses using the formulations of the invention. Bothchemically modified and unmodified RNA vaccines are useful according tothe invention. Surprisingly, in contrast to prior art reports that itwas preferable to use chemically unmodified mRNA formulated in a carrierfor the production of vaccines, it is described herein that chemicallymodified mRNA-LNP vaccines required a much lower effective mRNA dosethan unmodified mRNA, i.e., tenfold less than unmodified mRNA whenformulated in carriers other than LNP. Both the chemically modified andunmodified RNA vaccines of the invention produce better immune responsesthan mRNA vaccines formulated in a different lipid carrier.

In other aspects the invention encompasses a method of treating anelderly subject age 60 years or older comprising administering to thesubject a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a respiratoryvirus antigenic polypeptide in an effective amount to vaccinate thesubject.

In other aspects the invention encompasses a method of treating a youngsubject age 17 years or younger comprising administering to the subjecta nucleic acid vaccine comprising one or more RNA polynucleotides havingan open reading frame encoding a respiratory virus antigenic polypeptidein an effective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating an adultsubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a respiratory virus antigenic polypeptide in an effectiveamount to vaccinate the subject.

In some aspects the invention is a method of vaccinating a subject witha combination vaccine including at least two nucleic acid sequencesencoding respiratory antigens wherein the dosage for the vaccine is acombined therapeutic dosage wherein the dosage of each individualnucleic acid encoding an antigen is a sub therapeutic dosage. In someembodiments, the combined dosage is 25 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments, the combined dosage is 100 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments the combined dosage is 50 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments, the combined dosage is 75 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments, the combined dosage is 150 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments, the combined dosage is 400 micrograms of the RNApolynucleotide in the nucleic acid vaccine administered to the subject.In some embodiments, the sub therapeutic dosage of each individualnucleic acid encoding an antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 micrograms. In other embodimentsthe nucleic acid vaccine is chemically modified and in other embodimentsthe nucleic acid vaccine is not chemically modified.

The RNA polynucleotide is one of SEQ ID NO: 1-4, 9-12, 20-23, 35-46,57-61, and 64-80 and includes at least one chemical modification. Inother embodiments the RNA polynucleotide is one of SEQ ID NO: 1-4, 9-12,20-23, 35-46, 57-61, and 64-80 and does not include any nucleotidemodifications, or is unmodified. In yet other embodiments the at leastone RNA polynucleotide encodes an antigenic protein of any of SEQ ID NO:5-8, 12-13, 24-34, and 47-50 and includes at least one chemicalmodification. In other embodiments the RNA polynucleotide encodes anantigenic protein of any of SEQ ID NO: 5-8, 12-13, 24-34, and 47-50 anddoes not include any nucleotide modifications, or is unmodified.

In preferred aspects, vaccines of the invention (e.g., LNP-encapsulatedmRNA vaccines) produce prophylactically- and/ortherapeutically-efficacious levels, concentrations and/or titers ofantigen-specific antibodies in the blood or serum of a vaccinatedsubject. As defined herein, the term antibody titer refers to the amountof antigen-specific antibody produces in s subject, e.g., a humansubject. In exemplary embodiments, antibody titer is expressed as theinverse of the greatest dilution (in a serial dilution) that still givesa positive result. In exemplary embodiments, antibody titer isdetermined or measured by enzyme-linked immunosorbent assay (ELISA). Inexemplary embodiments, antibody titer is determined or measured byneutralization assay, e.g., by microneutralization assay. In certainaspects, antibody titer measurement is expressed as a ratio, such as1:40, 1:100, etc.

In exemplary embodiments of the invention, an efficacious vaccineproduces an antibody titer of greater than 1:40, greater that 1:100,greater than 1:400, greater than 1:1000, greater than 1:2000, greaterthan 1:3000, greater than 1:4000, greater than 1:500, greater than1:6000, greater than 1:7500, greater than 1:10000. In exemplaryembodiments, the antibody titer is produced or reached by 10 daysfollowing vaccination, by 20 days following vaccination, by 30 daysfollowing vaccination, by 40 days following vaccination, or by 50 ormore days following vaccination. In exemplary embodiments, the titer isproduced or reached following a single dose of vaccine administered tothe subject. In other embodiments, the titer is produced or reachedfollowing multiple doses, e.g., following a first and a second dose(e.g., a booster dose.)

In exemplary aspects of the invention, antigen-specific antibodies aremeasured in units of μg/ml or are measured in units of IU/L(International Units per liter) or mIU/ml (milli International Units perml). In exemplary embodiments of the invention, an efficacious vaccineproduces >0.5 μg/ml, >0.1 μg/ml, >0.2 μg/ml, >0.35 μg/ml, >0.5 μg/ml, >1μg/ml, >2 μg/ml, >5 μg/ml or >10 μg/ml. In exemplary embodiments of theinvention, an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml. Inexemplary embodiments, the antibody level or concentration is producedor reached by 10 days following vaccination, by 20 days followingvaccination, by 30 days following vaccination, by 40 days followingvaccination, or by 50 or more days following vaccination. In exemplaryembodiments, the level or concentration is produced or reached followinga single dose of vaccine administered to the subject. In otherembodiments, the level or concentration is produced or reached followingmultiple doses, e.g., following a first and a second dose (e.g., abooster dose.) In exemplary embodiments, antibody level or concentrationis determined or measured by enzyme-linked immunosorbent assay (ELISA).In exemplary embodiments, antibody level or concentration is determinedor measured by neutralization assay, e.g., by microneutralization assay.

The details of various embodiments of the disclosure are set forth inthe description below. Other features, objects, and advantages of thedisclosure will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of thedisclosure, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of thedisclosure.

FIG. 1 shows a schematic of one example of a RNA (e.g. mRNA) vaccineconstruct of the present disclosure. The construct depicts a humanMetapneumovirus and human respiratory syncytial virus full length fusionprotein obtained from wild-type strains (The Journal of GeneralVirology. 2008; 89(Pt 12): 3113-3118, incorporated herein by reference).

FIGS. 2A-2C are graphs showing the levels of anti-hMPV fusionprotein-specific antibodies in the serum of mice immunized with hMPVmRNA vaccines on day 0 (FIG. 2A), day 14 (FIG. 2B) and day 35 (FIG. 2C)post immunization. The mice were immunized with a single dose (2 μg or10 μg) on day 0 and were given a boost dose (2 μg or 10 μg) on day 21,hMPV fusion protein-specific antibodies were detected at up to 1:10000dilution of serum on day 35 for both doses.

FIGS. 3A-3C are graphs showing the result of IgG isotyping in the serumof mice immunized with hMPV mRNA vaccines. The levels of hMPV fusionprotein-specific IgG2a (FIG. 3A) and IgG1 (FIG. 3B) antibodies in theserum are measured by ELISA. FIG. 3C shows that hMPV fusion protein mRNAvaccine induced a mixed Th1/Th2 cytokine response with a Th1 bias.

FIG. 4 is a graph showing in vitro neutralization of a hMPV B2 strain(TN/91-316) using the sera of mice immunized with a mRNA vaccineencoding hMPV fusion protein. Mouse serum obtained from mice receiving a10 μg or a 2 μg dose contained hMPV-neutralizing antibodies.

FIGS. 5A-5C are graphs showing a Th1 cytokine response induced by a hMPVfusion peptide pool (15-mers-50 (overlap)) in splenocytes isolated frommice immunized with the hMPV mRNA vaccines. Virus-free media was used asa negative control and Concanavalin A (ConA, a positive control forsplenocyte stimulation) was included. The cytokines tested includedIFN-γ (FIG. 5A), IL-2 (FIG. 5B) and IL12 (FIG. 5C).

FIGS. 6A-6E are graphs showing the Th2 cytokine response induced by ahMPV fusion peptide pool (15-mers-50) in splenocytes isolated from miceimmunized with the hMPV mRNA vaccines. Virus-free media was used as anegative control and Concanavalin A was also included. The cytokinestested included IL-10 (FIG. 6A), TNF-α (FIG. 6B), IL4 (FIG. 6C), IL-5(FIG. 6D) and IL-6 (FIG. 6E).

FIGS. 7A-7C are graphs showing the Th1 response induced by inactivatedhMPV virus in splenocytes isolated from mice immunized with hMPV mRNAvaccines. Virus-free media was used as a negative control andConcanavalin A was included. The cytokines tested included IFN-γ (FIG.7A), IL-2 (FIG. 7B) and IL12 (FIG. 7C).

FIGS. 8A-8E are graphs showing the Th2 response induced by inactivatedhMPV virus in splenocytes isolated from mice immunized with the hMPVmRNA vaccines. Virus-free media was used as a negative control andConcanavalin A was included. The cytokines tested include IL-10 (FIG.8A), TNF-α (FIG. 8B), IL4 (FIG. 8C), IL-5 (FIG. 8D) and IL-6 (FIG. 8E).

FIGS. 9A-9B are graphs showing the results of cotton rat challengeexperiments. Two different doses of the hMPV mRNA vaccines were used (2μg or 10 μg doses) to immunize the cotton rats before challenge. ThehMPV mRNA vaccines reduced the viral titer in the lung and nose of thecotton rat, with the 10 μg dose being more effective in reducing viraltiter. Use of a 10 μg dose resulted in 100% protection in the lung and a2 log reduction in nose viral titer. Use of a 2 μg dose resulted in a 1log reduction in lung vital titer and no reduction in nose viral titer.The vaccine was administered on Day 0, and a boost was administered onDay 21.

FIG. 10 is a graph showing the lung histopathology of cotton rats thatreceived hMPV mRNA vaccines. Pathology associated with vaccine-enhanceddisease was not observed in immunized groups.

FIG. 11 is a graph showing hMPV neutralization antibody titers in cottonrats that received hMPV mRNA vaccines (2 μg or 10 μg doses) on days 35and 42 post immunization.

FIG. 12 is a graph showing the lung and nose viral load in cotton ratschallenged with a hMPV/A2 strain after immunization with the indicatedmRNA vaccines (hMPV mRNA vaccine or hMPV/PIV mRNA combination vaccine).Vaccinated cotton rats showed reduced lung and nose viral loads afterchallenge, compared to control.

FIG. 13 is a graph showing the lung and nose viral load in cotton ratschallenged with PIV3 strain after immunization with indicated mRNAvaccines (PIV mRNA vaccine or hMPV/PIV combination vaccine). Vaccinatedcotton rats showed reduced lung and nose viral loads after challenge,compared to control.

FIG. 14 is a graph showing hMPV neutralizing antibody titers in cottonrats that received different dosages of hMPV mRNA vaccines or hMPV/PIVcombination mRNA vaccines on day 42 post immunization. The dosages ofthe vaccine are indicated in Table 9.

FIG. 15 is a graph showing PIV3 neutralizing antibody titers in cottonrats that received different dosages of PIV mRNA vaccines or hMPV/PIVcombination mRNA vaccines on day 42 post immunization. The dosages ofthe vaccine are indicated in Table 9.

FIG. 16 is a graph showing the lung histopathology score of cotton ratsimmunized with hMPV mRNA vaccines, PIV mRNA vaccines or hMPV/PIVcombination mRNA vaccines as indicated in Table 9. Low occurrence ofalevolitis and interstitial pneumonia was observed, indicating noantibody-dependent enhancement (ADE) of hMPV associated diseases.

FIG. 17 is a graph showing the reciprocal MERS-CoV neutralizing antibodytiters in mice immunized with Betacoronavirus mRNA vaccine encoding theMERS-CoV full-length Spike protein, on days 0, 21, 42, and 56 postimmunization.

FIG. 18 is a graph showing the reciprocal MERS-CoV neutralizing antibodytiters in mice immunized with Betacoronavirus mRNA vaccine encodingeither the MERS-CoV full-length Spike protein, or the S2 subunit of theSpike protein. The full length spike protein induced a stronger immuneresponse compared to the S2 subunit alone.

FIGS. 19A-19C are graphs showing the viral load in the nose and throat,the bronchoalveolar lavage (BAL), or the lungs of New Zealand whiterabbits 4 days post challenge with MERS-CoV. The New Zealand whiterabbits were immunized with one 20 μg-dose (on day 0) or two 20 μg-doses(on day 0 and 21) of MERS-CoV mRNA vaccine encoding the full-lengthSpike protein before challenge. FIG. 19A shows that two doses ofMERS-CoV mRNA vaccine resulted in a 3 log reduction of viral load in thenose and led to complete protection in the throat of the New Zealandwhite rabbits. FIG. 19B shows that two doses of MERS-CoV mRNA vaccineresulted in a 4 log reduction of viral load in the BAL of the NewZealand white rabbits. FIG. 19C show one dose of MERS-CoV mRNA vaccineresulted in a 2 log reduction of viral load, while two doses of MERS-CoVmRNA vaccine resulted in an over 4 log reduction of viral load in thelungs of the New Zealand white rabbits.

FIGS. 20A-20B are images and graphs showing viral load or replicatingvirus detected by PCR in the lungs of New Zealand white rabbits 4 dayspost challenge with MERS-CoV. The New Zealand white rabbits wereimmunized with a single 20 μg dose (on day 0, Group 1a) of MERS-CoV mRNAvaccine encoding the full-length Spike protein, two 20 μg doses (on day0 and 21, Group 1b) of MERS-CoV mRNA vaccine encoding the full-lengthSpike protein, or placebo (Group 2) before challenge. FIG. 20A showsthat two doses of 20 μg a MERS-CoV mRNA vaccine reduced over 99% (2 log)of viruses in the lungs of New Zealand white rabbits. FIG. 20B showsthat the group of New Zealand white rabbits that received 2 doses of 20μg MERS-CoV mRNA vaccine did not have any detectable replicatingMERS-CoV virus in their lungs.

FIG. 21 is a graph showing the MERS-CoV neutralizing antibody titers inNew Zealand white rabbits immunized with MERS-CoV mRNA vaccine encodingthe full-length Spike protein. Immunization of the in New Zealand whiterabbits were carried out as described in FIGS. 21A-21C. The results showthat two doses of 20 μg MERS-CoV mRNA vaccine induced a significantamount of neutralizing antibodies against MERS-CoV (EC₅₀ between500-1000). The MERS-CoV mRNA vaccine induced antibody titer is 3-5 foldbetter than any other vaccines tested in the same model.

DETAILED DESCRIPTION

The present disclosure provides, in some embodiments, vaccines thatcomprise RNA (e.g., mRNA) polynucleotides encoding a humanMetapneumovirus (hMPV) antigenic polypeptide, a parainfluenza virus type3 (PIV3) antigenic polypeptide, a respiratory syncytial virus (RSV)antigenic polypeptide, a measles virus (MeV) antigenic polypeptide, or aBetacoronavirus antigenic polypeptide (e.g., Middle East respiratorysyndrome coronavirus (MERS-CoV), SARS-CoV, human coronavirus(HCoV)-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH (New Haven) andHCoV-HKU1) (see, e.g., Esper F. et al. Emerging Infectious Diseases,12(5), 2006; and Pyrc K. et al. Journal of Virology, 81(7):3051-57,2007, the contents of each of which is here incorporated by reference intheir entirety). The present disclosure also provides, in someembodiments, combination vaccines that comprise at least one RNA (e.g.,mRNA) polynucleotide encoding at least two antigenic polypeptidesselected from hMPV antigenic polypeptides, PIV3 antigenic polypeptides,RSV antigenic polypeptides, MeV antigenic polypeptides and BetaCoVantigenic polypeptides. Also provided herein are methods ofadministering the RNA (e.g., mRNA) vaccines, methods of producing theRNA (e.g., mRNA) vaccines, compositions (e.g., pharmaceuticalcompositions) comprising the RNA (e.g., mRNA) vaccines, and nucleicacids (e.g., DNA) encoding the RNA (e.g., mRNA) vaccines. In someembodiments, a RNA (e.g., mRNA) vaccine comprises an adjuvant, such as aflagellin adjuvant, as provided herein.

The RNA (e.g., mRNA) vaccines (e.g., hMPV, PIV3, RSV, MeV, BetaCoV RNAvaccines and combinations thereof), in some embodiments, may be used toinduce a balanced immune response, comprising both cellular and humoralimmunity, without many of the risks associated with DNA vaccination.

The entire contents of International Application No. PCT/US2015/02740 isincorporated herein by reference.

Human Metapneumovirus (hMPV)

hMPV shares substantial homology with respiratory syncytial virus (RSV)in its surface glycoproteins. hMPV fusion protein (F) is related toother paramyxovirus fusion proteins and appears to have homologousregions that may have similar functions. The hMPV fusion protein aminoacid sequence contains features characteristic of other paramyxovirus Fproteins, including a putative cleavage site and potential N-linkedglycosylation sites. Paramyxovirus fusion proteins are synthesized asinactive precursors (F0) that are cleaved by host cell proteases intothe biologically fusion-active F1 and F2 domains (see, e.g., Cseke G. etal. Journal of Virology 2007; 81(2):698-707, incorporated herein byreference). hMPV has one putative cleavage site, in contrast to the twosites established for RSV F, and only shares 34% amino acid sequenceidentity with RSV F. F2 is extracellular and disulfide linked to F1.Fusion proteins are type I glycoproteins existing as trimers, with two4-3 heptad repeat domains at the N- and C-terminal regions of theprotein (HR1 and HR2), which form coiled-coil alpha-helices. Thesecoiled coils become apposed in an antiparallel fashion when the proteinundergoes a conformational change into the fusogenic state. There is ahydrophobic fusion peptide N proximal to the N-terminal heptad repeat,which is thought to insert into the target cell membrane, while theassociation of the heptad repeats brings the transmembrane domain intoclose proximity, inducing membrane fusion (see, e.g., Baker, K A et al.Mol. Cell 1999; 3:309-319). This mechanism has been proposed for anumber of different viruses, including RSV, influenza virus, and humanimmunodeficiency virus. Fusion proteins are major antigenic determinantsfor all known paramyxoviruses and for other viruses that possess similarfusion proteins such as human immunodeficiency virus, influenza virus,and Ebola virus.

In some embodiments, a hMPV vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding hMPV fusion protein (F). Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding a F1 or F2 subunit of a hMPV Fprotein. In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding hMPV glycoprotein(G). In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding hMPV matrix protein(M). In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding hMPV phosphoprotein(P). In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding hMPV nucleoprotein(N). In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding hMPV SH protein(SH).

In some embodiments, a hMPV vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein, G protein, Mprotein, P protein, N protein and SH protein.

In some embodiments, a hMPV vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein and G protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and M protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and P protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and N protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and SH protein.

In some embodiments, a hMPV vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding G protein and M protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and P protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and N protein. Insome embodiments, a hMPV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and SH protein.

In some embodiments, a hMPV vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein, G protein and Mprotein. In some embodiments, a hMPV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding F protein, Gprotein and P protein. In some embodiments, a hMPV vaccine of thepresent disclosure comprises a RNA (e.g., mRNA) polynucleotide encodingF protein, G protein and N protein. In some embodiments, a hMPV vaccineof the present disclosure comprises a RNA (e.g., mRNA) polynucleotideencoding F protein, G protein and SH protein.

A hMPV vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide having an open reading frame encoding at least one hMPVantigenic polypeptide identified by any one of SEQ ID NO: 5-8 (Table 3;see also amino acid sequences of Table 4).

A hMPV vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide encoded by a nucleic acid (e.g., DNA) identified by anyone of SEQ ID NO: 1-4 (Table 2).

The present disclosure is not limited by a particular strain of hMPV.The strain of hMPV used in a vaccine may be any strain of hMPV.Non-limiting examples of strains of hMPV for use as provide hereininclude the CAN98-75 (CAN75) and the CAN97-83 (CAN83) hMPV strains(Skiadopoulos M H et al. J Virol. 20014; 78(13)6927-37, incorporatedherein by reference), a hMPV A1, A2, B1 or B2 strain (see, e.g., deGraaf M et al. The Journal of General Virology 2008; 89:975-83; Peret TC T et al. The Journal of Infectious Disease 2002; 185:1660-63,incorporated herein by reference), a hMPV isolate TN/92-4 (e.g., SEQ IDNO: 1 and 5), a hMPV isolate NL/1/99 (e.g., SEQ ID NO: 2 and 6), or ahMPV isolate PER/CFI0497/2010/B (e.g., SEQ ID NO: 3 and 7).

In some embodiments, at least one hMPV antigenic polypeptide is obtainedfrom a hMPV A1, A2, B1 or B2 strain (see, e.g., de Graaf M et al. TheJournal of General Virology 2008; 89:975-83; Peret T C T et al. TheJournal of Infectious Disease 2002; 185:1660-63, incorporated herein byreference). In some embodiments, at least one antigenic polypeptide isobtained from the CAN98-75 (CAN75) hMPV strain. In some embodiments, atleast one antigenic polypeptide is obtained from the CAN97-83 (CAN83)hMPV strain. In some embodiments, at least one antigenic polypeptide isobtained from hMPV isolate TN/92-4 (e.g., SEQ ID NO: 1 and 5). In someembodiments, at least one antigenic polypeptide is obtained from hMPVisolate NL/1/99 (e.g., SEQ ID NO: 2 and 6). In some embodiments, atleast one antigenic polypeptide is obtained from hMPV isolatePER/CFI0497/2010/B (e.g., SEQ ID NO: 3 and 7).

In some embodiments, hMPV vaccines comprise RNA (e.g., mRNA)polynucleotides encoding a hMPV antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith hMPV F protein and having F protein activity.

A protein is considered to have F protein activity if, for example, theprotein acts to fuse the viral envelope and host cell plasma membrane,mediates viral entry into a host cell via an interaction witharginine-glycine-aspartate RGD-binding integrins, or a combinationthereof (see, e.g., Cox R G et al. J Virol. 2012; 88(22):12148-60,incorporated herein by reference).

In some embodiments, hMPV vaccines comprise RNA (e.g., mRNA)polynucleotides encoding hMPV antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith hMPV G protein and having G protein activity.

A protein is considered to have G protein activity if, for example, theprotein acts to modulate (e.g., inhibit) hMPV-induced cellular (immune)responses (see, e.g., Bao X et al. PLoS Pathog. 2008; 4(5):e1000077,incorporated herein by reference).

Human parainfluenza virus type 3 (PIV3)

Parainfluenza viruses belong to the family Paramyxoviridae. These areenveloped viruses with a negative-sense single-stranded RNA genome.Parainfluenza viruses belong to the subfamily Paramyxoviridae, which issubdivided into three genera: Respirovirus (PIV-1, PIV-3, and Sendaivirus (SeV)), Rubulavirus (PIV-2, PIV-4 and mumps virus) andMorbillivirus (measles virus, rinderpest virus and canine distempervirus (CDV)). Their genome, a ˜15 500 nucleotide-long negative-sense RNAmolecule, encodes two envelope glycoproteins, thehemagglutinin-neuraminidase (HN), the fusion protein (F or F0), which iscleaved into F1 and F2 subunits, a matrix protein (M), a nucleocapsidprotein (N) and several nonstructural proteins including the viralreplicase (L). All parainfluenza viruses, except for PIV-1, express anon-structural V protein that blocks IFN signaling in the infected celland acts therefore as a virulence factor (see, e.g., Nishio M et al. JVirol. 2008; 82(13):6130-38).

PIV3 hemagglutinin-neuraminidase (HN), a structural protein, is found onthe viral envelope, where it is necessary for attachment and cell entry.It recognizes and binds to sialic acid-containing receptors on the hostcell's surface. As a neuroaminidase, HN removes sialic acid from virusparticles, preventing self-aggregation of the virus, and promoting theefficient spread of the virus. Furthermore, HN promotes the activity ofthe fusion (F or F0) protein, contributing to the penetration of thehost cell's surface.

PIV3 fusion protein (PIV3 F) is located on the viral envelope, where itfacilitates the viral fusion and cell entry. The F protein is initiallyinactive, but proteolytic cleavage leads to its active forms, F1 and F2,which are linked by disulfide bonds. This occurs when the HN proteinbinds its receptor on the host cell's surface. During early phases ofinfection, the F glycoprotein mediates penetration of the host cell byfusion of the viral envelope to the plasma membrane. In later stages ofthe infection, the F protein facilitates the fusion of the infectedcells with neighboring uninfected cells, which leads to the formation ofa syncytium and spread of the infection.

PIV3 matrix protein (M) is found within the viral envelope and assistswith viral assembly. It interacts with the nucleocapsid and envelopeglycoproteins, where it facilitates the budding of progeny virusesthrough its interactions with specific sites on the cytoplasmic tail ofthe viral glycoproteins and nucleocapsid. It also plays a role intransporting viral components to the budding site.

PIV3 phosphoprotein (P) and PIV3 large polymerase protein (L) are foundin the nucleocapsid where they form part of the RNA polymerase complex.The L protein, a viral RNA-dependent RNA polymerase, facilitates genomictranscription, while the host cell's ribosomes translate the viral mRNAinto viral proteins.

PIV3 V is a non-structural protein that blocks IFN signaling in theinfected cell, therefore acting as a virulence factor.

PIV3 nucleoprotein (N) encapsidates the genome in a ratio of 1 N per 6ribonucleotides, protecting it from nucleases. The nucleocapsid (NC) hasa helical structure. The encapsidated genomic RNA is termed the NC andserves as template for transcription and replication. Duringreplication, encapsidation by PIV3 N is coupled to RNA synthesis and allreplicative products are resistant to nucleases. PIV3 Nhomo-multimerizes to form the nucleocapsid and binds to viral genomicRNA. PIV3 N binds the P protein and thereby positions the polymerase onthe template.

In some embodiments, a PIV3 vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding PIV3 fusion protein (F). Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding a F1 or F2 subunit of a PIV3 Fprotein. In some embodiments, a PIV3 vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding PIV3hemagglutinin-neuraminidase (HN) (see, e.g., van Wyke Coelingh K L etal. J Virol. 1987; 61(5):1473-77, incorporated herein by reference). Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding PIV3 matrix protein (M). Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding PIV3 phosphoprotein (P). Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding PIV3 nucleoprotein (N).

In some embodiments, a PIV3 vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein, HN protein, Mprotein, P protein, and N protein.

In some embodiments, a PIV3 vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein and HN protein. Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and M protein. Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and P protein. Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and N protein.

In some embodiments, a PIV3 vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding HN protein and M protein. Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HN protein and P protein. Insome embodiments, a PIV3 vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HN protein and N protein.

In some embodiments, a PIV3 vaccine of the present disclosure comprisesa RNA (e.g., mRNA) polynucleotide encoding F protein, HN protein and Mprotein. In some embodiments, a PIV3 vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding F protein, HNprotein and P protein. In some embodiments, a PIV3 vaccine of thepresent disclosure comprises a RNA (e.g., mRNA) polynucleotide encodingF protein, HN protein and N protein.

A PIV3 vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide having an open reading frame encoding at least one PIV3antigenic polypeptide identified by any one of SEQ ID NO: 12-13 (Table6; see also amino acid sequences of Table 7).

A PIV3 vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide encoded by a nucleic acid (e.g., DNA) identified by anyone of SEQ ID NO: 9-12 (Table 5; see also nucleic acid sequences ofTable 7).

The present disclosure is not limited by a particular strain of PIV3.The strain of PIV3 used in a vaccine may be any strain of PIV3. Anon-limiting example of a strain of PIV3 for use as provide hereinincludes HPIV3/Homo sapiens/PER/FLA4815/2008.

In some embodiments, PIV3 vaccines comprise RNA (e.g., mRNA)polynucleotides encoding a PIV3 antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith PIV3 F protein and having F protein activity.

In some embodiments, PIV3 vaccines comprise RNA (e.g., mRNA)polynucleotides encoding PIV3 antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith PIV3 hemagglutinin-neuraminidase (HN) and havinghemagglutinin-neuraminidase activity.

A protein is considered to have hemagglutinin-neuraminidase activity if,for example, it is capable of both receptor binding and receptorcleaving. Such proteins are major surface glycoproteins that havefunctional sites for cell attachment and for neuraminidase activity.They are able to cause red blood cells to agglutinate and to cleave theglycosidic linkages of neuraminic acids, so they have the potential toboth bind a potential host cell and then release the cell if necessary,for example, to prevent self-aggregation of the virus.

In some embodiments, PIV3 vaccines comprise RNA (e.g., mRNA)polynucleotides encoding PIV3 antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith PIV3 HN, F (e.g., F, F1 or F2), M, N, L or V and having HN, F(e.g., F, F1 or F2), M, N, L or V activity, respectively.

Respiratory Syncytial Virus (RSV)

RSV is a negative-sense, single-stranded RNA virus of the genusPneumovirinae. The virus is present in at least two antigenic subgroups,known as Group A and Group B, primarily resulting from differences inthe surface G glycoproteins. Two RSV surface glycoproteins—G andF—mediate attachment with and attachment to cells of the respiratoryepithelium. F surface glycoproteins mediate coalescence of neighboringcells. This results in the formation of syncytial cells. RSV is the mostcommon cause of bronchiolitis. Most infected adults develop mildcold-like symptoms such as congestion, low-grade fever, and wheezing.Infants and small children may suffer more severe symptoms such asbronchiolitis and pneumonia. The disease may be transmitted among humansvia contact with respiratory secretions.

The genome of RSV encodes at least three surface glycoproteins,including F, G, and SH, four nucleocapsid proteins, including L, P, N,and M2, and one matrix protein, M. Glycoprotein F directs viralpenetration by fusion between the virion and the host membrane.Glycoprotein G is a type II transmembrane glycoprotein and is the majorattachment protein. SH is a short integral membrane protein. Matrixprotein M is found in the inner layer of the lipid bilayer and assistsvirion formation. Nucleocapsid proteins L, P, N, and M2 modulatereplication and transcription of the RSV genome. It is thought thatglycoprotein G tethers and stabilizes the virus particle at the surfaceof bronchial epithelial cells, while glycoprotein F interacts withcellular glycosaminoglycans to mediate fusion and delivery of the RSVvirion contents into the host cell (Krzyzaniak M A et al. PLoS Pathog2013; 9(4)).

In some embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein. In some embodiments,a PIV3 vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding G protein. In some embodiments, a PIV3 vaccineof the present disclosure comprises a RNA (e.g., mRNA) polynucleotideencoding L protein. In some embodiments, a PIV3 vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Pprotein. In some embodiments, a PIV3 vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding N protein. In someembodiments, a PIV3 vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide encoding M2 protein. In some embodiments, aPIV3 vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding M protein.

In some embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein, G protein, Lprotein, P protein, N protein, M2 protein and M protein.

In some embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and G protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and L protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and P protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and N protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and M2 protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and M protein.

In some embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and L protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and P protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and N protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and M2 protein. Insome embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding G protein and M protein.

In some embodiments, a RSV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein, G protein and Lprotein. In some embodiments, a RSV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding F protein, Gprotein and P protein. In some embodiments, a RSV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Fprotein, G protein and N protein. In some embodiments, a RSV vaccine ofthe present disclosure comprises a RNA (e.g., mRNA) polynucleotideencoding F protein, G protein and M2 protein. In some embodiments, a RSVvaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding F protein, G protein and M protein.

The present disclosure is not limited by a particular strain of RSV. Thestrain of RSV used in a vaccine may be any strain of RSV.

In some embodiments, RSV vaccines comprise RNA (e.g., mRNA)polynucleotides encoding a RSV antigenic polypeptides having at least95%, at least 96%, at least 97%, at least 98% or at least 99% identitywith RSV F protein and having F protein activity.

In some embodiments, RSV vaccines comprise RNA (e.g., mRNA)polynucleotides encoding RSV antigenic polypeptides having at least 95%,at least 96%, at least 97%, at least 98% or at least 99% identity withRSV G protein and having G protein activity.

A protein is considered to have G protein activity if, for example, theprotein acts to modulate (e.g., inhibit) hMPV-induced cellular (immune)responses (see, e.g., Bao X et al. PLoS Pathog. 2008; 4(5):e1000077,incorporated herein by reference).

Measles Virus (MeV)

Molecular epidemiologic investigations and virologic surveillancecontribute notably to the control and prevention of measles. Nearly halfof measles-related deaths worldwide occur in India, yet virologicsurveillance data are incomplete for many regions of the country.Previous studies have documented the presence of measles virus genotypesD4, D7, and D8 in India, and genotypes D5, D9, D11, H1, and G3 have beendetected in neighboring countries. Recently, MeV genotype B3 wasdetected in India (Kuttiatt V S et al. Emerg Infect Dis. 2014; 20(10):1764-66).

The glycoprotein complex of paramyxoviruses mediates receptor bindingand membrane fusion. In particular, the MeV fusion (F) protein executesmembrane fusion, after receptor binding by the hemagglutinin (HA)protein (Muhlebach M D et al. Journal of Virology 2008;82(22):11437-45). The MeV P gene codes for three proteins: P, anessential polymerase cofactor, and V and C, which have multiplefunctions but are not strictly required for viral propagation incultured cells. V shares the amino-terminal domain with P but has azinc-binding carboxyl-terminal domain, whereas C is translated from anoverlapping reading frame. The MeV C protein is an infectivity factor.During replication, the P protein binds incoming monomeric nucleocapsid(N) proteins with its amino-terminal domain and positions them forassembly into the nascent ribonucleocapsid. The P protein amino-terminaldomain is natively unfolded (Deveaux P et al. Journal of Virology 2004;78(21):11632-40).

In some embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein. In someembodiments, a MeV vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide encoding F protein. In some embodiments, aMeV vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding P protein. In some embodiments, a MeV vaccine ofthe present disclosure comprises a RNA (e.g., mRNA) polynucleotideencoding V protein. In some embodiments, a MeV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Cprotein.

In some embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein, F protein, Pprotein, V protein and C protein.

In some embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein and F protein. Insome embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein and P protein. Insome embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein and V protein. Insome embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein and C protein.

some embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and P protein. Insome embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and V protein. Insome embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding F protein and C protein.

In some embodiments, a MeV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding HA protein, F protein and Pprotein. In some embodiments, a MeV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding HA protein, Fprotein and V protein. In some embodiments, a MeV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding HAprotein, F protein and C protein.

In some embodiments, MeV vaccines comprise RNA (e.g., mRNA) encoding aMeV antigenic polypeptide having at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity with MeV HA protein andhaving MeV HA protein activity.

In some embodiments, MeV vaccines comprise RNA (e.g., mRNA) encoding aMeV antigenic polypeptide having at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity with MeV F protein and havingMeV F protein activity.

A protein is considered to have HA protein activity if the proteinmediates receptor binding and/or membrane fusion. MeV F protein executesmembrane fusion, after receptor binding by the MeV HA protein.

A MeV vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide having an open reading frame encoding at least one MeVantigenic polypeptide identified by any one of SEQ ID NO: 47-50 (Table14; see also amino acid sequences of Table 15).

A MeV vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide identified by any one of SEQ ID NO: 37, 40, 43, 46 (Table13).

A MeV vaccine may comprise, for example, at least one RNA (e.g., mRNA)polynucleotide encoded by a nucleic acid (e.g., DNA) identified by anyone of SEQ ID NO: 35, 36, 38, 39, 41, 42, 44 and 45 (Table 13).

The present disclosure is not limited by a particular strain of MeV. Thestrain of MeV used in a vaccine may be any strain of MeV. Non-limitingexamples of strains of MeV for use as provide herein include B3/B3.1,C2, D4, D6, D7, D8, G3, H1, Moraten, Rubeovax, MVi/New Jersey.USA/45.05,MVi/Texas.USA/4.07, AIK-C, MVi/New York.USA/26.09/3,MVi/California.USA/16.03, MVi/Virginia.USA/15.09,MVi/California.USA/8.04, and MVi/Pennsylvania.USA/20.09.

MeV proteins may be from MeV genotype D4, D5, D7, D8, D9, D11, H1, G3 orB3. In some embodiments, a MeV HA protein or a MeV F protein is from MeVgenotype D8. In some embodiments, a MeV HA protein or a MeV F protein isfrom MeV genotype B3.

Betacoronaviruses (BetaCoV)

MERS-Co V. MERS-CoV is a positive-sense, single-stranded RNA virus ofthe genus Betacoronavirus. The genomes are phylogenetically classifiedinto two clades, clade A and clade B. It has a strong tropism fornon-ciliated bronchial epithelial cells, evades the innate immuneresponse and antagonizes interferon (IFN) production in infected cells.Dipeptyl peptidase 4 (DDP4, also known as CD26) has been identified as afunctional cellular receptor for MERS-CoV. Its enzymatic activity is notrequired for infection, although its amino acid sequence is highlyconserved across species and is expressed in the human bronchialepithelium and kidneys. Most infected individuals develop severe acuterespiratory illnesses, including fever, cough, and shortness of breath,and the virus can be fatal. The disease may be transmitted among humans,generally among those in close contact.

The genome of MERS-CoV encodes at least four unique accessory proteins,such as 3, 4a, 4b and 5, two replicase proteins (open reading frame 1aand 1b), and four major structural proteins, including spike (S),envelope (E), nucleocapsid (N), and membrane (M) proteins (Almazan F etal. MBio 2013; 4(5):e00650-13). The accessory proteins play nonessentialroles in MERS-CoV replication, but they are likely structural proteinsor interferon antagonists, modulating in vivo replication efficiencyand/or pathogenesis, as in the case of SARS-CoV (Almazan F et al. MBio2013; 4(5):e00650-13; Totura A L et al. Curr Opin Virol 2012;2(3):264-75; Scobey T et al. Proc Natl Acad Sci USA 2013;110(40):16157-62). The other proteins of MERS-CoV maintain differentfunctions in virus replication. The E protein, for example, involves invirulence, and deleting the E-coding gene results inreplication-competent and propagation-defective viruses or attenuatedviruses (Almazan F et al. MBio 2013; 4(5):e00650-13). The S protein isparticularly essential in mediating virus binding to cells expressingreceptor dipeptidyl peptidase-4 (DPP4) through receptor-binding domain(RBD) in the S1 subunit, whereas the S2 subunit subsequently mediatesvirus entry via fusion of the virus and target cell membranes (Li F. JVirol 2015; 89(4):1954-64; Raj V S et al. Nature 2013; 495(7440):251-4).

In some embodiments, a MERS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein. In someembodiments, a MERS-CoV vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding the S1 subunit of the Sprotein. In some embodiments, a MERS-CoV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding the S2subunit of the S protein. In some embodiments, a MERS-CoV vaccine of thepresent disclosure comprises a RNA (e.g., mRNA) polynucleotide encodingE protein. In some embodiments, a MERS-CoV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Nprotein. In some embodiments, a MERS-CoV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Mprotein.

In some embodiments, a MERS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2), E protein, N protein and M protein.

In some embodiments, a MERS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2) and E protein. In some embodiments, a MERS-CoV vaccine of thepresent disclosure comprises a RNA (e.g., mRNA) polynucleotide encodingS protein (S, S1 and/or S2) and N protein. In some embodiments, aMERS-CoV vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding S protein (S, S1 and/or S2) and M protein.

In some embodiments, a MERS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2), E protein and M protein. In some embodiments, a MERS-CoVvaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding S protein (S, S1 and/or S2), E protein and Nprotein. In some embodiments, a MERS-CoV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Sprotein (S, S1 and/or S2), M protein and N protein. In some embodiments,a MERS-CoV vaccine of the present disclosure comprises a RNA (e.g.,mRNA) polynucleotide encoding E protein, M protein and N protein.

A MERS-CoV vaccine may comprise, for example, at least one RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding at least oneMERS-CoV antigenic polypeptide identified by any one of SEQ ID NO: 24-38or 33 (Table 11; see also amino acid sequences of Table 12).

A MERS-CoV vaccine may comprise, for example, at least one RNA (e.g.,mRNA) polynucleotide encoded by a nucleic acid (e.g., DNA) identified byany one of SEQ ID NO: 20-23 (Table 10).

The present disclosure is not limited by a particular strain ofMERS-CoV. The strain of MERS-CoV used in a vaccine may be any strain ofMERS-CoV. Non-limiting examples of strains of MERS-CoV for use asprovide herein include Riyadh_14_2013, and 2cEMC/2012, Hasa_1_2013.

SARS-CoV. The genome of SARS-CoV includes of a single, positive-strandRNA that is approximately 29,700 nucleotides long. The overall genomeorganization of SARS-CoV is similar to that of other coronaviruses. Thereference genome includes 13 genes, which encode at least 14 proteins.Two large overlapping reading frames (ORFs) encompass 71% of the genome.The remainder has 12 potential ORFs, including genes for structuralproteins S (spike), E (small envelope), M (membrane), and N(nucleocapsid). Other potential ORFs code for unique putativeSARS-CoV-specific polypeptides that lack obvious sequence similarity toknown proteins. A detailed analysis of the SARS-CoV genome has beenpublished in J Mol Biol 2003; 331: 991-1004.

In some embodiments, a SARS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2), E protein, N protein and M protein.

In some embodiments, a SARS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2) and E protein. In some embodiments, a SARS-CoV vaccine of thepresent disclosure comprises a RNA (e.g., mRNA) polynucleotide encodingS protein (S, S1 and/or S2) and N protein. In some embodiments, aSARS-CoV vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding S protein (S, S1 and/or S2) and M protein.

In some embodiments, a SARS-CoV vaccine of the present disclosurecomprises a RNA (e.g., mRNA) polynucleotide encoding S protein (S, S1and/or S2), E protein and M protein. In some embodiments, a SARS-CoVvaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding S protein (S, S1 and/or S2), E protein and Nprotein. In some embodiments, a SARS-CoV vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding Sprotein (S, S1 and/or S2), M protein and N protein. In some embodiments,a SARS-CoV vaccine of the present disclosure comprises a RNA (e.g.,mRNA) polynucleotide encoding E protein, M protein and N protein.

A SARS-CoV vaccine may comprise, for example, at least one RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding at least oneSARS-CoV antigenic polypeptide identified by any one of SEQ ID NO: 29,32 or 34 (Table 11; see also amino acid sequences of Table 12).

The present disclosure is not limited by a particular strain ofSARS-CoV. The strain of SARS-CoV used in a vaccine may be any strain ofSARS-CoV.

HCoV-OC43. Human coronavirus OC43 is an enveloped, positive-sense,single-stranded RNA virus in the species Betacoronavirus-1 (genusBetacoronavirus, subfamily Coronavirinae, family Coronaviridae, orderNidovirales). Four HCoV-OC43 genotypes (A to D), have been identifiedwith genotype D most likely arising from recombination. The completegenome sequencing of two genotype C and D strains and bootscan analysisshows recombination events between genotypes B and C in the generationof genotype D. Of 29 strains identified, none belong to the more ancientgenotype A. Along with HCoV-229E, a species in the Alphacoronavirusgenus, HCoV-OC43 are among the known viruses that cause the common cold.Both viruses can cause severe lower respiratory tract infections,including pneumonia in infants, the elderly, and immunocompromisedindividuals such as those undergoing chemotherapy and those withHIV-AIDS.

HCoV-HKU1. Human coronavirus HKU1 (HCoV-HKU1) is a positive-sense,single-stranded RNA virus with the HE gene, which distinguishes it as agroup 2, or Betacoronavirus. It was discovered in January 2005 in twopatients in Hong Kong. The genome of HCoV-HKU1 is a 29,926-nucleotide,polyadenylated RNA. The GC content is 32%, the lowest among all knowncoronaviruses. The genome organization is the same as that of othergroup II coronaviruses, with the characteristic gene order 1a, 1b, HE,S, E, M, and N. Furthermore, accessory protein genes are present betweenthe S and E genes (ORF4) and at the position of the N gene (ORF8). TheTRS is presumably located within the AAUCUAAAC sequence, which precedeseach ORF except E. As in sialodacryoadenitis virus and mouse hepatitisvirus (MHV), translation of the E protein possibly occurs via aninternal ribosomal entry site. The 3′ untranslated region contains apredicted stem-loop structure immediately downstream of the N ORF(nucleotide position 29647 to 29711). Further downstream, a pseudoknotstructure is present at nucleotide position 29708 to 29760. Both RNAstructures are conserved in group II coronaviruses and are critical forvirus replication.

HCoV-NL63. The RNA genome of human coronavirus NL63 (HCoV-NL63) is27,553 nucleotides, with a poly(A) tail (FIG. 1). With a GC content of34%, HCoV-NL63 has one of the lowest GC contents of the coronaviruses,for which GC content ranges from 32 to 42%. Untranslated regions of 286and 287 nucleotides are present at the 5′ and 3′ termini, respectively.Genes predicted to encode the S, E, M, and N proteins are found in the3′ part of the HCoV-NL63 genome. The HE gene, which is present in somegroup II coronaviruses, is absent, and there is only a single,monocistronic accessory protein ORF (ORF3) located between the S and Egenes. Subgenomic mRNAs are generated for all ORFs (S, ORF3, E, M, andN), and the core sequence of the TRS of HCoV-NL63 is defined as AACUAAA.This sequence is situated upstream of every ORF except for the E ORF,which contains the suboptimal core sequence AACUAUA. Interestingly, a13-nucleotide sequence with perfect homology to the leader sequence issituated upstream of the suboptimal E TRS. Annealing of this13-nucleotide sequence to the leader sequence may act as a compensatorymechanism for the disturbed leader-TRS/body-TRS interaction.

HCoV-229E. Human coronavirus 229E (HCoV-229E) is a single-stranded,positive-sense, RNA virus species in the Alphacoronavirus genus of thesubfamily Coronavirinae, in the family Coronaviridae, of the orderNidovirales. Along with Human coronavirus OC43, it is responsible forthe common cold. HCoV-NL63 and HCoV-229E are two of the four humancoronaviruses that circulate worldwide. These two viruses are unique intheir relationship towards each other. Phylogenetically, the viruses aremore closely related to each other than to any other human coronavirus,yet they only share 65% sequence identity. Moreover, the viruses usedifferent receptors to enter their target cell. HCoV-NL63 is associatedwith croup in children, whereas all signs suggest that the virusprobably causes the common cold in healthy adults. HCoV-229E is a provencommon cold virus in healthy adults, so it is probable that both virusesinduce comparable symptoms in adults, even though their mode ofinfection differs (HCoV-NL63 and HCoV-229E are two of the four humancoronaviruses that circulate worldwide. These two viruses are unique intheir relationship towards each other. Phylogenetically, the viruses aremore closely related to each other than to any other human coronavirus,yet they only share 65% sequence identity. Moreover, the viruses usedifferent receptors to enter their target cell. HCoV-NL63 is associatedwith croup in children, whereas all signs suggest that the virusprobably causes the common cold in healthy adults. HCoV-229E is a provencommon cold virus in healthy adults, so it is probable that both virusesinduce comparable symptoms in adults, even though their mode ofinfection differs (Dijkman R. et al. J Formos Med Assoc. 2009 April;108(4):270-9, the contents of which is incorporated herein by referencein their entirety).

Combination Vaccines

Embodiments of the present disclosure also provide combination RNA(e.g., mRNA) vaccines. A “combination RNA (e.g., mRNA) vaccine” of thepresent disclosure refers to a vaccine comprising at least one (e.g., atleast 2, 3, 4, or 5) RNA (e.g., mRNA) polynucleotide having an openreading frame encoding a combination of any two or more (or all of)antigenic polypeptides selected from hMPV antigenic polypeptides, PIV3antigenic polypeptides, RSV antigenic polypeptides, MeV antigenicpolypeptides, and BetaCoV antigenic polypeptides (e.g., selected fromMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide, a RSV antigenic polypeptide, a MeV antigenicpolypeptide, and a BetaCoV antigenic polypeptide (e.g., selected fromMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptideand a PIV3 antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptideand a RSV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptideand a MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptideand a BetaCoV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptideand a RSV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptideand a MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptideand a BetaCoV antigenic polypeptide (e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a RSV antigenic polypeptide anda MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a RSV antigenic polypeptide anda BetaCoV antigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a MeV antigenic polypeptide anda BetaCoV antigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide, a RSV antigenic polypeptide and a MeVantigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide, a RSV antigenic polypeptide and a BetaCoVantigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide, a MeV antigenic polypeptide and a BetaCoVantigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aRSV antigenic polypeptide, a MeV antigenic polypeptide and a BetaCoVantigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptide, aRSV antigenic polypeptide, a MeV antigenic polypeptide and a BetaCoVantigenic polypeptide (e.g., selected from MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide and a RSV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide and a MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aPIV3 antigenic polypeptide and a BetaCoV antigenic polypeptide (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aRSV antigenic polypeptide and a MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aRSV antigenic polypeptide and a BetaCoV antigenic polypeptide (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV antigenic polypeptide, aMeV antigenic polypeptide and a BetaCoV antigenic polypeptide (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptide, aRSV antigenic polypeptide and a MeV antigenic polypeptide.

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a PIV3 antigenic polypeptide, aRSV antigenic polypeptide and a BetaCoV antigenic polypeptide (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1).

In some embodiments, a combination RNA (e.g., mRNA) vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a RSV antigenic polypeptide, aMeV antigenic polypeptide and a BetaCoV antigenic polypeptide (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1).

Other combination respiratory virus RNA (e.g., mRNA) vaccines areencompassed by the present disclosure.

It has been discovered that the mRNA vaccines described herein aresuperior to current vaccines in several ways. First, the lipidnanoparticle (LNP) delivery is superior to other formulations includinga protamine base approach described in the literature and no additionaladjuvants are to be necessary. The use of LNPs enables the effectivedelivery of chemically modified or unmodified mRNA vaccines.Additionally it has been demonstrated herein that both modified andunmodified LNP formulated mRNA vaccines were superior to conventionalvaccines by a significant degree. In some embodiments the mRNA vaccinesof the invention are superior to conventional vaccines by a factor of atleast 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000fold.

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines have not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the invention a class of formulations fordelivering mRNA vaccines in vivo that results in significantly enhanced,and in many respects synergistic, immune responses including enhancedantigen generation and functional antibody production withneutralization capability. These results can be achieved even whensignificantly lower doses of the mRNA are administered in comparisonwith mRNA doses used in other classes of lipid based formulations. Theformulations of the invention have demonstrated significant unexpectedin vivo immune responses sufficient to establish the efficacy offunctional mRNA vaccines as prophylactic and therapeutic agents.Additionally, self-replicating RNA vaccines rely on viral replicationpathways to deliver enough RNA to a cell to produce an immunogenicresponse. The formulations of the invention do not require viralreplication to produce enough protein to result in a strong immuneresponse. Thus, the mRNA of the invention are not self-replicating RNAand do not include components necessary for viral replication.

The invention involves, in some aspects, the surprising finding thatlipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA vaccines, including chemically modified andunmodified mRNA vaccines. The efficacy of mRNA vaccines formulated inLNP was examined in vivo using several distinct antigens. The resultspresented herein demonstrate the unexpected superior efficacy of themRNA vaccines formulated in LNP over other commercially availablevaccines.

In addition to providing an enhanced immune response, the formulationsof the invention generate a more rapid immune response with fewer dosesof antigen than other vaccines tested. The mRNA-LNP formulations of theinvention also produce quantitatively and qualitatively better immuneresponses than vaccines formulated in a different carriers.

The data described herein demonstrate that the formulations of theinvention produced significant unexpected improvements over existingantigen vaccines. Additionally, the mRNA-LNP formulations of theinvention are superior to other vaccines even when the dose of mRNA islower than other vaccines. Mice immunized with either 10 μg or 2 μgdoses of an hMPV fusion protein mRNA LNP vaccine or a PIV3 mRNA LNPvaccine produced neutralizing antibodies which for instance,successfully neutralized the hMPV B2 virus. A 10 μg dose of mRNA vaccineprotected 100% of mice from lethal challenge and drastically reduced theviral titer after challenge (˜2 log reduction).

Two 20 μg doses of MERS-CoV mRNA LNP vaccine significantly reduced viralload and induced significant amount of neutralizing antibodies againstMERS-CoV (EC₅₀ between 500-1000). The MERS-CoV mRNA vaccine inducedantibody titer was 3-5 fold better than any other vaccines tested in thesame model.

The LNP used in the studies described herein has been used previously todeliver siRNA in various animal models as well as in humans. In view ofthe observations made in association with the siRNA delivery of LNPformulations, the fact that LNP is useful in vaccines is quitesurprising. It has been observed that therapeutic delivery of siRNAformulated in LNP causes an undesirable inflammatory response associatedwith a transient IgM response, typically leading to a reduction inantigen production and a compromised immune response. In contrast to thefindings observed with siRNA, the LNP-mRNA formulations of the inventionare demonstrated herein to generate enhanced IgG levels, sufficient forprophylactic and therapeutic methods rather than transient IgMresponses.

Nucleic Acids/Polynucleotides

Respiratory virus vaccines, as provided herein, comprise at least one(one or more) ribonucleic acid (RNA) (e.g., mRNA) polynucleotide havingan open reading frame encoding at least one antigenic polypeptideselected from hMPV, PIV3, RSV, MeV and BetaCoV (e.g., selected fromMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand HCoV-HKU1) antigenic polypeptides. The term “nucleic acid” includesany compound and/or substance that comprises a polymer of nucleotides(nucleotide monomer). These polymers are referred to as polynucleotides.Thus, the terms “nucleic acid” and “polynucleotide” are usedinterchangeably.

Nucleic acids may be or may include, for example, ribonucleic acids(RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs),glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), lockednucleic acids (LNAs, including LNA having a β-D-ribo configuration,α-LNA having an α-L-ribo configuration (a diastereomer of LNA),2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNAhaving a 2′-amino functionalization), ethylene nucleic acids (ENA),cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo. The skilled artisan will appreciatethat, except where otherwise noted, polynucleotide sequences set forthin the instant application will recite “T”s in a representative DNAsequence but where the sequence represents RNA (e.g., mRNA), the “T”swould be substituted for “U”s. Thus, any of the RNA polynucleotidesencoded by a DNA identified by a particular sequence identificationnumber may also comprise the corresponding RNA (e.g., mRNA) sequenceencoded by the DNA, where each “T” of the DNA sequence is substitutedwith “U.”

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features, which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics.

In some embodiments, a RNA polynucleotide of an RNA (e.g., mRNA) vaccineencodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antigenicpolypeptides. In some embodiments, a RNA (e.g., mRNA) polynucleotide ofa respiratory virus vaccine encodes at least 10, 20, 30, 40, 50, 60, 70,80, 90 or 100 antigenic polypeptides. In some embodiments, a RNA (e.g.,mRNA) polynucleotide of a respiratory virus vaccine encodes at least 100or at least 200 antigenic polypeptides. In some embodiments, a RNApolynucleotide of an respiratory virus vaccine encodes 1-10, 5-15,10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or2-100 antigenic polypeptides.

Polynucleotides of the present disclosure, in some embodiments, arecodon optimized. Codon optimization methods are known in the art and maybe used as provided herein. Codon optimization, in some embodiments, maybe used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art—non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity, less than 90% sequence identity, less than 85%sequence identity, less than 80% sequence identity, or less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or antigenicpolypeptide)).

In some embodiments, a codon-optimized sequence shares between 65% and85% (e.g., between about 67% and about 85%, or between about 67% andabout 80%) sequence identity to a naturally-occurring sequence or awild-type sequence (e.g., a naturally-occurring or wild-type mRNAsequence encoding a polypeptide or protein of interest (e.g., anantigenic protein or polypeptide)). In some embodiments, acodon-optimized sequence shares between 65% and 75%, or about 80%sequence identity to a naturally-occurring sequence or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide)).

In some embodiments a codon-optimized RNA (e.g., mRNA) may, forinstance, be one in which the levels of G/C are enhanced. TheG/C-content of nucleic acid molecules may influence the stability of theRNA. RNA having an increased amount of guanine (G) and/or cytosine (C)residues may be functionally more stable than nucleic acids containing alarge amount of adenine (A) and thymine (T) or uracil (U) nucleotides.WO02/098443 discloses a pharmaceutical composition containing an mRNAstabilized by sequence modifications in the translated region. Due tothe degeneracy of the genetic code, the modifications work bysubstituting existing codons for those that promote greater RNAstability without changing the resulting amino acid. The approach islimited to coding regions of the RNA.

Antigens/Antigenic Polypeptides

In some embodiments, an antigenic polypeptide (e.g., a hMPV, PIV3, RSV,MeV or BetaCoV antigenic polypeptide) is longer than 25 amino acids andshorter than 50 amino acids. Polypeptides include gene products,naturally occurring polypeptides, synthetic polypeptides, homologs,orthologs, paralogs, fragments and other equivalents, variants, andanalogs of the foregoing. A polypeptide may be a single molecule or maybe a multi-molecular complex such as a dimer, trimer or tetramer.Polypeptides may also comprise single chain polypeptides or multichainpolypeptides, such as antibodies or insulin, and may be associated orlinked to each other. Most commonly, disulfide linkages are found inmultichain polypeptides. The term “polypeptide” may also apply to aminoacid polymers in which at least one amino acid residue is an artificialchemical analogue of a corresponding naturally-occurring amino acid.

A “polypeptide variant” is a molecule that differs in its amino acidsequence relative to a native sequence or a reference sequence. Aminoacid sequence variants may possess substitutions, deletions, insertions,or a combination of any two or three of the foregoing, at certainpositions within the amino acid sequence, as compared to a nativesequence or a reference sequence. Ordinarily, variants possess at least50% identity to a native sequence or a reference sequence. In someembodiments, variants share at least 80% identity or at least 90%identity with a native sequence or a reference sequence.

In some embodiments “variant mimics” are provided. A “variant mimic”contains at least one amino acid that would mimic an activated sequence.For example, glutamate may serve as a mimic for phosphoro-threonineand/or phosphoro-serine. Alternatively, variant mimics may result indeactivation or in an inactivated product containing the mimic. Forexample, phenylalanine may act as an inactivating substitution fortyrosine, or alanine may act as an inactivating substitution for serine.

“Orthologs” refers to genes in different species that evolved from acommon ancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution. Identification of orthologs isimportant for reliable prediction of gene function in newly sequencedgenomes.

“Analogs” is meant to include polypeptide variants that differ by one ormore amino acid alterations, for example, substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present disclosure provides several types of compositions that arepolynucleotide or polypeptide based, including variants and derivatives.These include, for example, substitutional, insertional, deletion andcovalent variants and derivatives. The term “derivative” is synonymouswith the term “variant” and generally refers to a molecule that has beenmodified and/or changed in any way relative to a reference molecule or astarting molecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. For example, sequence tags or amino acids, such as oneor more lysines, can be added to peptide sequences (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidedetection, purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalresidues or N-terminal residues) alternatively may be deleted dependingon the use of the sequence, as for example, expression of the sequenceas part of a larger sequence that is soluble, or linked to a solidsupport.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. Substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore (e.g., 3, 4 or 5) amino acids have been substituted in the samemolecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminiand any combination(s) thereof.

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” As used herein whenreferring to polynucleotides the terms “site” as it pertains tonucleotide based embodiments is used synonymously with “nucleotide.” Asite represents a position within a peptide or polypeptide orpolynucleotide that may be modified, manipulated, altered, derivatizedor varied within the polypeptide-based or polynucleotide-basedmolecules.

As used herein the terms “termini” or “terminus” when referring topolypeptides or polynucleotides refers to an extremity of a polypeptideor polynucleotide respectively. Such extremity is not limited only tothe first or final site of the polypeptide or polynucleotide but mayinclude additional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH2))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N- and C-termini.Alternatively, the termini of the polypeptides may be modified such thatthey begin or end, as the case may be, with a non-polypeptide basedmoiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein having a length of 10, 20,30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% identical to any of the sequences described herein can beutilized in accordance with the disclosure. In some embodiments, apolypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided herein or referenced herein. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 amino acids that are greater than 80%, 90%, 95%, or 100%identical to any of the sequences described herein, wherein the proteinhas a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than80%, 75%, 70%, 65% to 60% identical to any of the sequences describedherein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure mayshare a certain degree of sequence similarity or identity with thereference molecules (e.g., reference polypeptides or referencepolynucleotides), for example, with art-described molecules (e.g.,engineered or designed molecules or wild-type molecules). The term“identity,” as known in the art, refers to a relationship between thesequences of two or more polypeptides or polynucleotides, as determinedby comparing the sequences. In the art, identity also means the degreeof sequence relatedness between two sequences as determined by thenumber of matches between strings of two or more amino acid residues ornucleic acid residues. Identity measures the percent of identicalmatches between the smaller of two or more sequences with gap alignments(if any) addressed by a particular mathematical model or computerprogram (e.g., “algorithms”). Identity of related peptides can bereadily calculated by known methods. “% identity” as it applies topolypeptide or polynucleotide sequences is defined as the percentage ofresidues (amino acid residues or nucleic acid residues) in the candidateamino acid or nucleic acid sequence that are identical with the residuesin the amino acid sequence or nucleic acid sequence of a second sequenceafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent identity. Methods and computer programs forthe alignment are well known in the art. Identity depends on acalculation of percent identity but may differ in value due to gaps andpenalties introduced in the calculation. Generally, variants of aparticular polynucleotide or polypeptide have at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% but less than 100% sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art. Such tools for alignment include those of the BLASTsuite (Stephen F. Altschul, et al. (1997).” Gapped BLAST and PSI-BLAST:a new generation of protein database search programs,” Nucleic AcidsRes. 25:3389-3402). Another popular local alignment technique is basedon the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently, a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) was developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.Other tools are described herein, specifically in the definition of“identity” below.

As used herein, the term “homology” refers to the overall relatednessbetween polymeric molecules, e.g. between nucleic acid molecules (e.g.DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNAmolecules and/or RNA molecules) and/or polypeptide molecules) that sharea threshold level of similarity or identity determined by alignment ofmatching residues are termed homologous. Homology is a qualitative termthat describes a relationship between molecules and can be based uponthe quantitative similarity or identity. Similarity or identity is aquantitative term that defines the degree of sequence match between twocompared sequences. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). Two polynucleotide sequences are consideredhomologous if the polypeptides they encode are at least 50%, 60%, 70%,80%, 90%, 95%, or even 99% for at least one stretch of at least 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Twoprotein sequences are considered homologous if the proteins are at least50%, 60%, 70%, 80%, or 90% identical for at least one stretch of atleast 20 amino acids.

Homology implies that the compared sequences diverged in evolution froma common origin. The term “homolog” refers to a first amino acidsequence or nucleic acid sequence (e.g., gene (DNA or RNA) or proteinsequence) that is related to a second amino acid sequence or nucleicacid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteinsseparated by the event of speciation or to the relationship betweengenes and/or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolvedfrom a common ancestral gene (or protein) by speciation. Typically,orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new functions, even if these are related to theoriginal one.

The term “identity” refers to the overall relatedness between polymericmolecules, for example, between polynucleotide molecules (e.g. DNAmolecules and/or RNA molecules) and/or between polypeptide molecules.Calculation of the percent identity of two polynucleic acid sequences,for example, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Incertain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleic acidsequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleic acid sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleic acid sequencescan, alternatively, be determined using the GAP program in the GCGsoftware package using an NWSgapdna.CMP matrix. Methods commonlyemployed to determine percent identity between sequences include, butare not limited to those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Multiprotein and Multicomponent Vaccines

The present disclosure encompasses respiratory virus vaccines comprisingmultiple RNA (e.g., mRNA) polynucleotides, each encoding a singleantigenic polypeptide, as well as respiratory virus vaccines comprisinga single RNA polynucleotide encoding more than one antigenic polypeptide(e.g., as a fusion polypeptide). Thus, a vaccine composition comprisinga RNA (e.g., mRNA) polynucleotide having an open reading frame encodinga first antigenic polypeptide and a RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding a second antigenic polypeptideencompasses (a) vaccines that comprise a first RNA polynucleotideencoding a first antigenic polypeptide and a second RNA polynucleotideencoding a second antigenic polypeptide, and (b) vaccines that comprisea single RNA polynucleotide encoding a first and second antigenicpolypeptide (e.g., as a fusion polypeptide). RNA (e.g., mRNA) vaccinesof the present disclosure, in some embodiments, comprise 2-10 (e.g., 2,3, 4, 5, 6, 7, 8, 9 or 10), or more, RNA polynucleotides having an openreading frame, each of which encodes a different antigenic polypeptide(or a single RNA polynucleotide encoding 2-10, or more, differentantigenic polypeptides). The antigenic polypeptides may be selected fromhMPV, PIV3, RSV, MEV and BetaCoV (e.g., selected from MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH andHCoV-HKU1) antigenic polypeptides.

In some embodiments, a respiratory virus vaccine comprises a RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding a viralcapsid protein, a RNA (e.g., mRNA) polynucleotide having an open readingframe encoding a viral premembrane/membrane protein, and a RNA (e.g.,mRNA) polynucleotide having an open reading frame encoding a viralenvelope protein. In some embodiments, a respiratory virus vaccinecomprises a RNA (e.g., mRNA) polynucleotide having an open reading frameencoding a viral fusion (F) protein and a RNA polynucleotide having anopen reading frame encoding a viral major surface glycoprotein (Gprotein). In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a viral F protein.In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a viral G protein.In some embodiments, a vaccine comprises a RNA (e.g., mRNA)polynucleotide having an open reading frame encoding a HN protein.

In some embodiments, a multicomponent vaccine comprises at least one RNA(e.g., mRNA) polynucleotide encoding at least one antigenic polypeptidefused to a signal peptide (e.g., any one of SEQ ID NO: 15-19). Thesignal peptide may be fused at the N-terminus or the C-terminus of anantigenic polypeptide. An antigenic polypeptide fused to a signalpeptide may be selected from hMPV, PIV3, RSV, MEV and BetaCoV (e.g.,selected from MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and HCoV-HKU1) antigenic polypeptides.

Signal Peptides

In some embodiments, antigenic polypeptides encoded by respiratory virusRNA (e.g., mRNA) polynucleotides comprise a signal peptide. Signalpeptides, comprising the N-terminal 15-60 amino acids of proteins, aretypically needed for the translocation across the membrane on thesecretory pathway and, thus, universally control the entry of mostproteins both in eukaryotes and prokaryotes to the secretory pathway.Signal peptides generally include three regions: an N-terminal region ofdiffering length, which usually comprises positively charged aminoacids; a hydrophobic region; and a short carboxy-terminal peptideregion. In eukaryotes, the signal peptide of a nascent precursor protein(pre-protein) directs the ribosome to the rough endoplasmic reticulum(ER) membrane and initiates the transport of the growing peptide chainacross it for processing. ER processing produces mature proteins,wherein the signal peptide is cleaved from precursor proteins, typicallyby a ER-resident signal peptidase of the host cell, or they remainuncleaved and function as a membrane anchor. A signal peptide may alsofacilitate the targeting of the protein to the cell membrane. The signalpeptide, however, is not responsible for the final destination of themature protein. Secretory proteins devoid of additional address tags intheir sequence are by default secreted to the external environment.During recent years, a more advanced view of signal peptides hasevolved, showing that the functions and immunodominance of certainsignal peptides are much more versatile than previously anticipated.

Respiratory virus vaccines of the present disclosure may comprise, forexample, RNA (e.g., mRNA) polynucleotides encoding an artificial signalpeptide, wherein the signal peptide coding sequence is operably linkedto and is in frame with the coding sequence of the antigenicpolypeptide. Thus, respiratory virus vaccines of the present disclosure,in some embodiments, produce an antigenic polypeptide comprising anantigenic polypeptide (e.g., hMPV, PIV3, RSV, MeV or BetaCoV) fused to asignal peptide. In some embodiments, a signal peptide is fused to theN-terminus of the antigenic polypeptide. In some embodiments, a signalpeptide is fused to the C-terminus of the antigenic polypeptide.

In some embodiments, the signal peptide fused to the antigenicpolypeptide is an artificial signal peptide. In some embodiments, anartificial signal peptide fused to the antigenic polypeptide encoded bythe RNA (e.g., mRNA) vaccine is obtained from an immunoglobulin protein,e.g., an IgE signal peptide or an IgG signal peptide. In someembodiments, a signal peptide fused to the antigenic polypeptide encodedby a RNA (e.g., mRNA) vaccine is an Ig heavy chain epsilon-1 signalpeptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS (SEQ IDNO: 16). In some embodiments, a signal peptide fused to the antigenicpolypeptide encoded by the (e.g., mRNA) RNA (e.g., mRNA) vaccine is anIgGk chain V-III region HAH signal peptide (IgGk SP) having the sequenceof METPAQLLFLLLLWLPDTTG (SEQ ID NO: 15). In some embodiments, the signalpeptide is selected from: Japanese encephalitis PRM signal sequence(MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 17), VSVg protein signal sequence(MKCLLYLAFLFIGVNCA; SEQ ID NO: 18) and Japanese encephalitis JEV signalsequence (MWLVSLAIVTACAGA; SEQ ID NO: 19).

In some embodiments, the antigenic polypeptide encoded by a RNA (e.g.,mRNA) vaccine comprises an amino acid sequence identified by any one ofSEQ ID NO: 5-8, 12-13, 24-34, 47-50 or 54-56 (Tables 3, 6, 11, 14 or 17;see also amino acid sequences of Tables 4, 7, 12 or 15) fused to asignal peptide identified by any one of SEQ ID NO: 15-19 (Table 8). Theexamples disclosed herein are not meant to be limiting and any signalpeptide that is known in the art to facilitate targeting of a protein toER for processing and/or targeting of a protein to the cell membrane maybe used in accordance with the present disclosure.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide has a length of20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55,25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50,35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40,20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30,25-30, 15-25, 20-25, or 15-20 amino acids.

A signal peptide is typically cleaved from the nascent polypeptide atthe cleavage junction during ER processing. The mature antigenicpolypeptide produce by a respiratory virus RNA (e.g., mRNA) vaccine ofthe present disclosure typically does not comprise a signal peptide.

Chemical Modifications

Respiratory virus vaccines of the present disclosure, in someembodiments, comprise at least RNA (e.g. mRNA) polynucleotide having anopen reading frame encoding at least one antigenic polypeptide thatcomprises at least one chemical modification.

The terms “chemical modification” and “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides inat least one of their position, pattern, percent or population.Generally, these terms do not refer to the ribonucleotide modificationsin naturally occurring 5′-terminal mRNA cap moieties. With respect to apolypeptide, the term “modification” refers to a modification relativeto the canonical set 20 amino acids. Polypeptides, as provided herein,are also considered “modified” of they contain amino acid substitutions,insertions or a combination of substitutions and insertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Modifications of polynucleotides include, without limitation, thosedescribed herein. Polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) may comprise modifications that arenaturally-occurring, non-naturally-occurring or the polynucleotide maycomprise a combination of naturally-occurring andnon-naturally-occurring modifications. Polynucleotides may include anyuseful modification, for example, of a sugar, a nucleobase, or aninternucleoside linkage (e.g., to a linking phosphate, to aphosphodiester linkage or to the phosphodiester backbone).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on an internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphdioester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker may be incorporated into polynucleotides of thepresent disclosure.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the vaccines of the presentdisclosure include, but are not limited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine;2′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6,N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adeno sine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyo sine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguano sine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methoxyuridine; 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyaceticacid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2(thio)pseudouracil; 2′ deoxy uridine; 2′ fluorouridine; 2-(thio)uracil;2,4-(dithio)psuedouracil; 2′ methyl, 2′amino, 2′azido,2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP;2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxy uridine; 2′fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridineTP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of pseudouridine (ψ), N1-methylpseudouridine (m¹ψ),N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides) include a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of 1-methyl-pseudouridine (m¹ψ), 5-methoxy-uridine(mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine (ψ), α-thio-guanosine andα-thio-adenosine. In some embodiments, polynucleotides includes acombination of at least two (e.g., 2, 3, 4 or more) of theaforementioned modified nucleobases.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine(m⁵C). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ). Insome embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise 1-methyl-pseudouridine (m¹ψ) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine(s²U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2-thiouridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine(mo⁵U). In some embodiments, polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo⁵U) and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyluridine. In some embodiments polynucleotides (e.g., RNA polynucleotides,such as mRNA polynucleotides) comprise 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A). In some embodiments, polynucleotides (e.g.,RNA polynucleotides, such as mRNA polynucleotides) compriseN6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m⁵C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m⁵C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Exemplary nucleobases and In some embodiments, a modified nucleobase isa modified cytosine. nucleosides having a modified uridine include5-cyano uridine, and 4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), andN6-methyl-adenosine (m6A).

In some embodiments, a modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in apolynucleotide of the disclosure, or in a given predetermined sequenceregion thereof (e.g., in the mRNA including or excluding the polyAtail). In some embodiments, all nucleotides X in a polynucleotide of thepresent disclosure (or in a given sequence region thereof) are modifiednucleotides, wherein X may any one of nucleotides A, G, U, C, or any oneof the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C orA+G+C.

The polynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). Any remaining percentage isaccounted for by the presence of unmodified A, G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the polynucleotides may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the polynucleotide is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). n some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe polynucleotide is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Thus, in some embodiments, the RNA (e.g., mRNA) vaccines comprise a5′UTR element, an optionally codon optimized open reading frame, and a3′UTR element, a poly(A) sequence and/or a polyadenylation signalwherein the RNA is not chemically modified.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Urn), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-0H-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formylcytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethylcytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethylcytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂ Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-0H-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶ ₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate)(Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine,2′-F-ara-adenosine, 2′-F-adenosine, 2′-0H-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G±),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂ G), N2,7-dimethyl-guano sine (m^(2,7)G),N2,N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂ Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

N-Linked Glycosylation Site Mutants

N-linked glycans of viral proteins play important roles in modulatingthe immune response. Glycans can be important for maintaining theappropriate antigenic conformations, shielding potential neutralizationepitopes, and may alter the proteolytic susceptibility of proteins. Someviruses have putative N-linked glycosylation sites. Deletion ormodification of an N-linked glycosylation site may enhance the immuneresponse. Thus, the present disclosure provides, in some embodiments,RNA (e.g., mRNA) vaccines comprising nucleic acids (e.g., mRNA) encodingantigenic polypeptides that comprise a deletion or modification at oneor more N-linked glycosylation sites.

In Vitro Transcription of RNA (e.g., mRNA)

Respiratory virus vaccines of the present disclosure comprise at leastone RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, forexample, is transcribed in vitro from template DNA, referred to as an“in vitro transcription template.” In some embodiments, an in vitrotranscription template encodes a 5′ untranslated (UTR) region, containsan open reading frame, and encodes a 3′ UTR and a polyA tail. Theparticular nucleic acid sequence composition and length of an in vitrotranscription template will depend on the mRNA encoded by the template.

A “5′ untranslated region” (5′UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (3′UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides.

Flagellin Adjuvants

Flagellin is an approximately 500 amino acid monomeric protein thatpolymerizes to form the flagella associated with bacterial motion.Flagellin is expressed by a variety of flagellated bacteria (Salmonellatyphimurium for example) as well as non-flagellated bacteria (such asEscherichia coli). Sensing of flagellin by cells of the innate immunesystem (dendritic cells, macrophages, etc.) is mediated by the Toll-likereceptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf andNaip5. TLRs and NLRs have been identified as playing a role in theactivation of innate immune response and adaptive immune response. Assuch, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellinpolypeptides are publicly available in the NCBI GenBank database. Theflagellin sequences from S. Typhimurium, H. Pylori, V. Cholera, S.marcesens, S. flexneri, T. Pallidum, L. pneumophila, B. burgdorferei, C.difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.Mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli,among others are known.

A flagellin polypeptide, as used herein, refers to a full lengthflagellin protein, immunogenic fragments thereof, and peptides having atleast 50% sequence identify to a flagellin protein or immunogenicfragments thereof. Exemplary flagellin proteins include flagellin fromSalmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium(A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonellacholeraesuis (Q6V2X8), and SEQ ID NO: 54-56 (Table 17). In someembodiments, the flagellin polypeptide has at least 60%, 70%, 75%, 80%,90%, 95%, 97%, 98%, or 99% sequence identify to a flagellin protein orimmunogenic fragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenicfragment. An immunogenic fragment is a portion of a flagellin proteinthat provokes an immune response. In some embodiments, the immuneresponse is a TLR5 immune response. An example of an immunogenicfragment is a flagellin protein in which all or a portion of a hingeregion has been deleted or replaced with other amino acids. For example,an antigenic polypeptide may be inserted in the hinge region. Hingeregions are the hypervariable regions of a flagellin. Hinge regions of aflagellin are also referred to as “D3 domain or region, “propellerdomain or region,” “hypervariable domain or region” and “variable domainor region.” “At least a portion of a hinge region,” as used herein,refers to any part of the hinge region of the flagellin, or the entiretyof the hinge region. In other embodiments an immunogenic fragment offlagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment offlagellin.

The flagellin monomer is formed by domains D0 through D3. D0 and D1,which form the stem, are composed of tandem long alpha helices and arehighly conserved among different bacteria. The D1 domain includesseveral stretches of amino acids that are useful for TLR5 activation.The entire D1 domain or one or more of the active regions within thedomain are immunogenic fragments of flagellin. Examples of immunogenicregions within the D1 domain include residues 88-114 and residues411-431 (in Salmonella typhimurium FliC flagellin. Within the 13 aminoacids in the 88-100 region, at least 6 substitutions are permittedbetween Salmonella flagellin and other flagellins that still preserveTLR5 activation. Thus, immunogenic fragments of flagellin includeflagellin like sequences that activate TLR5 and contain a 13 amino acidmotif that is 53% or more identical to the Salmonella sequence in 88-100of FliC (LQRVRELAVQSAN; SEQ ID NO: 84).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA thatencodes a fusion protein of flagellin and one or more antigenicpolypeptides. A “fusion protein” as used herein, refers to a linking oftwo components of the construct. In some embodiments, a carboxy-terminusof the antigenic polypeptide is fused or linked to an amino terminus ofthe flagellin polypeptide. In other embodiments, an amino-terminus ofthe antigenic polypeptide is fused or linked to a carboxy-terminus ofthe flagellin polypeptide. The fusion protein may include, for example,one, two, three, four, five, six or more flagellin polypeptides linkedto one, two, three, four, five, six or more antigenic polypeptides. Whentwo or more flagellin polypeptides and/or two or more antigenicpolypeptides are linked such a construct may be referred to as a“multimer.”

Each of the components of a fusion protein may be directly linked to oneanother or they may be connected through a linker. For instance, thelinker may be an amino acid linker. The amino acid linker encoded for bythe RNA (e.g., mRNA) vaccine to link the components of the fusionprotein may include, for instance, at least one member selected from thegroup consisting of a lysine residue, a glutamic acid residue, a serineresidue and an arginine residue. In some embodiments the linker is 1-30,1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

In other embodiments the RNA (e.g., mRNA) vaccine includes at least twoseparate RNA polynucleotides, one encoding one or more antigenicpolypeptides and the other encoding the flagellin polypeptide. The atleast two RNA polynucleotides may be co-formulated in a carrier such asa lipid nanoparticle.

Broad Spectrum RNA (e.g., mRNA) Vaccines

There may be situations where persons are at risk for infection withmore than one strain of hMPV, PIV3, RSV, MeV and/or BetaCoV (includingMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand/or HCoV-HKU1). RNA (e.g., mRNA) therapeutic vaccines areparticularly amenable to combination vaccination approaches due to anumber of factors including, but not limited to, speed of manufacture,ability to rapidly tailor vaccines to accommodate perceived geographicalthreat, and the like. Moreover, because the vaccines utilize the humanbody to produce the antigenic protein, the vaccines are amenable to theproduction of larger, more complex antigenic proteins, allowing forproper folding, surface expression, antigen presentation, etc. in thehuman subject. To protect against more than one strain of hMPV, PIV3,RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1), a combinationvaccine can be administered that includes RNA (e.g., mRNA) encoding atleast one antigenic polypeptide protein (or antigenic portion thereof)of a first respiratory virus and further includes RNA encoding at leastone antigenic polypeptide protein (or antigenic portion thereof) of asecond respiratory virus. RNA (e.g., mRNA) can be co-formulated, forexample, in a single lipid nanoparticle (LNP) or can be formulated inseparate LNPs for co-administration.

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment ofrespiratory diseases/infections in humans and other mammals. Respiratoryvirus RNA (e.g. mRNA) vaccines can be used as therapeutic orprophylactic agents, alone or in combination with other vaccine(s). Theymay be used in medicine to prevent and/or treat respiratorydisease/infection. In exemplary aspects, the RNA (e.g., mRNA) vaccinesof the present disclosure are used to provide prophylactic protectionfrom hMPV, PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1).Prophylactic protection from hMPV, PIV3, RSV, MeV and/or BetaCoV(including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH and/or HCoV-HKU1) can be achieved following administration of aRNA (e.g., mRNA) vaccine of the present disclosure. Respiratory virusRNA (e.g., mRNA) vaccines of the present disclosure may be used to treator prevent viral “co-infections” containing two or more respiratoryinfections. Vaccines can be administered once, twice, three times, fourtimes or more, but it is likely sufficient to administer the vaccineonce (optionally followed by a single booster). It is possible, althoughless desirable, to administer the vaccine to an infected individual toachieve a therapeutic response. Dosing may need to be adjustedaccordingly.

A method of eliciting an immune response in a subject against hMPV,PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) is provided inaspects of the present disclosure. The method involves administering tothe subject a respiratory virus RNA (e.g., mRNA) vaccine comprising atleast one RNA (e.g., mRNA) polynucleotide having an open reading frameencoding at least one hMPV, PIV3, RSV, MeV and/or BetaCoV (includingMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand/or HCoV-HKU1) antigenic polypeptide thereof, thereby inducing in thesubject an immune response specific to hMPV, PIV3, RSV, MeV and/orBetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH and/or HCoV-HKU1) antigenic polypeptide or animmunogenic fragment thereof, wherein anti-antigenic polypeptideantibody titer in the subject is increased following vaccinationrelative to anti-antigenic polypeptide antibody titer in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine against hMPV, PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/orHCoV-HKU1). An “anti-antigenic polypeptide antibody” is a serum antibodythe binds specifically to the antigenic polypeptide.

In some embodiments, a RNA (e.g., mRNA) vaccine (e.g., a hMPV, PIV3,RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1 RNA vaccine)capable of eliciting an immune response is administered intramuscularlyvia a composition including a compound according to Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe) (e.g., Compound 3, 18, 20, 25,26, 29, 30, 60, 108-112, or 122).

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the RNA (e.g., mRNA) vaccines ofthe present disclosure. For instance, a traditional vaccine includes butis not limited to live/attenuated microorganism vaccines,killed/inactivated microorganism vaccines, subunit vaccines, proteinantigen vaccines, DNA vaccines, VLP vaccines, etc. In exemplaryembodiments, a traditional vaccine is a vaccine that has achievedregulatory approval and/or is registered by a national drug regulatorybody, for example the Food and Drug Administration (FDA) in the UnitedStates or the European Medicines Agency (EMA).

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log to 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against hMPV,PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1).

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log, 2 log, 3 log, 5 log or 10 log followingvaccination relative to anti-antigenic polypeptide antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against hMPV, PIV3, RSV, MeV and/or BetaCoV(including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH and/or HCoV-HKU1).

A method of eliciting an immune response in a subject against hMPV,PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) is provided inother aspects of the disclosure. The method involves administering tothe subject a respiratory virus RNA (e.g., mRNA) vaccine comprising atleast one RNA (e.g., mRNA) polynucleotide having an open reading frameencoding at least one hMPV, PIV3, RSV, MeV and/or BetaCoV (includingMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand/or HCoV-HKU1) antigenic polypeptide or an immunogenic fragmentthereof, thereby inducing in the subject an immune response specific tohMPV, PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1)antigenic polypeptide or an immunogenic fragment thereof, wherein theimmune response in the subject is equivalent to an immune response in asubject vaccinated with a traditional vaccine against the hMPV, PIV3,RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) at 2 times to100 times the dosage level relative to the RNA (e.g., mRNA) vaccine.

In some embodiments, the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at2, 3, 4, 5, 10, 50, 100 times the dosage level relative to the hMPV,PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) RNA (e.g.,mRNA) vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10-100 times, or 100-1000 times, the dosage level relative to the hMPV,PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) RNA (e.g.,mRNA) vaccine.

In some embodiments the immune response is assessed by determining[protein] antibody titer in the subject.

Some aspects of the present disclosure provide a method of eliciting animmune response in a subject against a In some embodiments the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine at 2, 3, 4, 5, 10, 50, 100 timesthe dosage level relative to the hMPV, PIV3, RSV, MeV and/or BetaCoV(including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH and/or HCoV-HKU1) RNA (e.g., mRNA) vaccine by administering tothe subject a respiratory virus RNA (e.g., mRNA) vaccine comprising atleast one RNA (e.g., mRNA) polynucleotide having an open reading frameencoding at least one hMPV, PIV3, RSV, MeV and/or BetaCoV (includingMERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NHand/or HCoV-HKU1) antigenic polypeptide, thereby inducing in the subjectan immune response specific to the antigenic polypeptide or animmunogenic fragment thereof, wherein the immune response in the subjectis induced 2 days to 10 weeks earlier relative to an immune responseinduced in a subject vaccinated with a prophylactically effective doseof a traditional vaccine against the hMPV, PIV3, RSV, MeV and/or BetaCoV(including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH and/or HCoV-HKU1). In some embodiments, the immune response inthe subject is induced in a subject vaccinated with a prophylacticallyeffective dose of a traditional vaccine at 2 times to 100 times thedosage level relative to the RNA (e.g., mRNA) vaccine.

In some embodiments, the immune response in the subject is induced 2days earlier, or 3 days earlier, relative to an immune response inducedin a subject vaccinated with a prophylactically effective dose of atraditional vaccine.

In some embodiments the immune response in the subject is induced 1week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to animmune response induced in a subject vaccinated with a prophylacticallyeffective dose of a traditional vaccine.

Also provided herein is a method of eliciting an immune response in asubject against hMPV, PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/orHCoV-HKU1) by administering to the subject a respiratory virus RNA(e.g., mRNA) vaccine having an open reading frame encoding a firstantigenic polypeptide, wherein the RNA polynucleotide does not include astabilization element, and wherein an adjuvant is not co-formulated orco-administered with the vaccine.

Therapeutic and Prophylactic Compositions

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention, treatment or diagnosis ofhMPV, PIV3, RSV, MeV and/or BetaCoV (including MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1) inhumans and other mammals, for example. Respiratory virus RNA (e.g. mRNA)vaccines can be used as therapeutic or prophylactic agents. They may beused in medicine to prevent and/or treat infectious disease. In someembodiments, the respiratory RNA (e.g., mRNA) vaccines of the presentdisclosure are used fin the priming of immune effector cells, forexample, to activate peripheral blood mononuclear cells (PBMCs) ex vivo,which are then infused (re-infused) into a subject.

In some embodiments, respiratory virus vaccine containing RNA (e.g.,mRNA) polynucleotides as described herein can be administered to asubject (e.g., a mammalian subject, such as a human subject), and theRNA (e.g., mRNA) polynucleotides are translated in vivo to produce anantigenic polypeptide.

The respiratory virus RNA (e.g., mRNA) vaccines may be induced fortranslation of a polypeptide (e.g., antigen or immunogen) in a cell,tissue or organism. In some embodiments, such translation occurs invivo, although such translation may occur ex vivo, in culture or invitro. In some embodiments, the cell, tissue or organism is contactedwith an effective amount of a composition containing a respiratory virusRNA (e.g., mRNA) vaccine that contains a polynucleotide that has atleast one a translatable region encoding an antigenic polypeptide.

An “effective amount” of an respiratory virus RNA (e.g. mRNA) vaccine isprovided based, at least in part, on the target tissue, target celltype, means of administration, physical characteristics of thepolynucleotide (e.g., size, and extent of modified nucleosides) andother components of the vaccine, and other determinants. In general, aneffective amount of the respiratory virus RNA (e.g., mRNA) vaccinecomposition provides an induced or boosted immune response as a functionof antigen production in the cell, preferably more efficient than acomposition containing a corresponding unmodified polynucleotideencoding the same antigen or a peptide antigen. Increased antigenproduction may be demonstrated by increased cell transfection (thepercentage of cells transfected with the RNA, e.g., mRNA, vaccine),increased protein translation from the polynucleotide, decreased nucleicacid degradation (as demonstrated, for example, by increased duration ofprotein translation from a modified polynucleotide), or altered antigenspecific immune response of the host cell.

In some embodiments, RNA (e.g. mRNA) vaccines (including polynucleotidestheir encoded polypeptides) in accordance with the present disclosuremay be used for treatment of hMPV, PIV3, RSV, MeV and/or BetaCoV(including MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH and/or HCoV-HKU1).

Respiratory RNA (e.g. mRNA) vaccines may be administeredprophylactically or therapeutically as part of an active immunizationscheme to healthy individuals or early in infection during theincubation phase or during active infection after onset of symptoms. Insome embodiments, the amount of RNA (e.g., mRNA) vaccine of the presentdisclosure provided to a cell, a tissue or a subject may be an amounteffective for immune prophylaxis.

Respiratory virus RNA (e.g. mRNA) vaccines may be administrated withother prophylactic or therapeutic compounds. As a non-limiting example,a prophylactic or therapeutic compound may be an adjuvant or a booster.As used herein, when referring to a prophylactic composition, such as avaccine, the term “booster” refers to an extra administration of theprophylactic (vaccine) composition. A booster (or booster vaccine) maybe given after an earlier administration of the prophylacticcomposition. The time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95years or more than 99 years. In some embodiments, the time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, respiratory virus RNA (e.g. mRNA) vaccines may beadministered intramuscularly or intradermally, similarly to theadministration of inactivated vaccines known in the art.

Respiratory virus RNA (e.g. mRNA) vaccines may be utilized in varioussettings depending on the prevalence of the infection or the degree orlevel of unmet medical need. As a non-limiting example, the RNA (e.g.,mRNA) vaccines may be utilized to treat and/or prevent a variety ofrespiratory infections. RNA (e.g., mRNA) vaccines have superiorproperties in that they produce much larger antibody titers and produceresponses early than commercially available anti-viralagents/compositions.

Provided herein are pharmaceutical compositions including respiratoryvirus RNA (e.g. mRNA) vaccines and RNA (e.g. mRNA) vaccine compositionsand/or complexes optionally in combination with one or morepharmaceutically acceptable excipients.

Respiratory virus RNA (e.g. mRNA) vaccines may be formulated oradministered alone or in conjunction with one or more other components.For instance, hMPV/PIV3/RSV RNA (e.g., mRNA) vaccines (vaccinecompositions) may comprise other components including, but not limitedto, adjuvants.

In some embodiments, respiratory virus (e.g. mRNA) vaccines do notinclude an adjuvant (they are adjuvant free).

Respiratory virus RNA (e.g. mRNA) vaccines may be formulated oradministered in combination with one or more pharmaceutically-acceptableexcipients. In some embodiments, vaccine compositions comprise at leastone additional active substances, such as, for example, atherapeutically-active substance, a prophylactically-active substance,or a combination of both. Vaccine compositions may be sterile,pyrogen-free or both sterile and pyrogen-free. General considerations inthe formulation and/or manufacture of pharmaceutical agents, such asvaccine compositions, may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, respiratory virus RNA (e.g. mRNA) vaccines areadministered to humans, human patients or subjects. For the purposes ofthe present disclosure, the phrase “active ingredient” generally refersto the RNA (e.g., mRNA) vaccines or the polynucleotides containedtherein, for example, RNA polynucleotides (e.g., mRNA polynucleotides)encoding antigenic polypeptides.

Formulations of the respiratory virus vaccine compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Respiratory virus RNA (e.g. mRNA) vaccines can be formulated using oneor more excipients to: (1) increase stability; (2) increase celltransfection; (3) permit the sustained or delayed release (e.g., from adepot formulation); (4) alter the biodistribution (e.g., target tospecific tissues or cell types); (5) increase the translation of encodedprotein in vivo; and/or (6) alter the release profile of encoded protein(antigen) in vivo. In addition to traditional excipients such as any andall solvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with respiratory virus RNA (e.g. mRNA) vaccines (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA (e.g., mRNA) vaccine may include one or morestabilizing elements. Stabilizing elements may include for instance ahistone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa proteinhas been identified. It is associated with the histone stem-loop at the3′-end of the histone messages in both the nucleus and the cytoplasm.Its expression level is regulated by the cell cycle; it peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA (e.g., mRNA) vaccines include a codingregion, at least one histone stem-loop, and optionally, a poly(A)sequence or polyadenylation signal. The poly(A) sequence orpolyadenylation signal generally should enhance the expression level ofthe encoded protein. The encoded protein, in some embodiments, is not ahistone protein, a reporter protein (e.g. Luciferase, GFP, EGFP,β-Galactosidase, EGFP), or a marker or selection protein (e.g.alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyltransferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, the RNA (e.g., mRNA) vaccine does not comprise ahistone downstream element (HDE). “Histone downstream element” (HDE)includes a purine-rich polynucleotide stretch of approximately 15 to 20nucleotides 3′ of naturally occurring stem-loops, representing thebinding site for the U7 snRNA, which is involved in processing ofhistone pre-mRNA into mature histone mRNA. Ideally, the inventivenucleic acid does not include an intron.

In some embodiments, the RNA (e.g., mRNA) vaccine may or may not containa enhancer and/or promoter sequence, which may be modified or unmodifiedor which may be activated or inactivated. In some embodiments, thehistone stem-loop is generally derived from histone genes, and includesan intramolecular base pairing of two neighbored partially or entirelyreverse complementary sequences separated by a spacer, including (e.g.,consisting of) a short sequence, which forms the loop of the structure.The unpaired loop region is typically unable to base pair with either ofthe stem loop elements. It occurs more often in RNA, as is a keycomponent of many RNA secondary structures, but may be present insingle-stranded DNA as well. Stability of the stem-loop structuregenerally depends on the length, number of mismatches or bulges, andbase composition of the paired region. In some embodiments, wobble basepairing (non-Watson-Crick base pairing) may result. In some embodiments,the at least one histone stem-loop sequence comprises a length of 15 to45 nucleotides.

In other embodiments the RNA (e.g., mRNA) vaccine may have one or moreAU-rich sequences removed. These sequences, sometimes referred to asAURES are destabilizing sequences found in the 3′UTR. The AURES may beremoved from the RNA (e.g., mRNA) vaccines. Alternatively the AURES mayremain in the RNA (e.g., mRNA) vaccine.

Nanoparticle Formulations

In some embodiments, respiratory virus RNA (e.g. mRNA) vaccines areformulated in a nanoparticle. In some embodiments, respiratory virus RNA(e.g. mRNA) vaccines are formulated in a lipid nanoparticle. In someembodiments, respiratory virus RNA (e.g. mRNA) vaccines are formulatedin a lipid-polycation complex, referred to as a cationic lipidnanoparticle. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In some embodiments,respiratory virus RNA (e.g., mRNA) vaccines are formulated in a lipidnanoparticle that includes a non-cationic lipid such as, but not limitedto, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

A lipid nanoparticle formulation may be influenced by, but not limitedto, the selection of the cationic lipid component, the degree ofcationic lipid saturation, the nature of the PEGylation, ratio of allcomponents and biophysical parameters such as size. In one example bySemple et al. (Nature Biotech. 2010 28:172-176), the lipid nanoparticleformulation is composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid canmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200).

In some embodiments, lipid nanoparticle formulations may comprise 35 to45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipidand/or 55% to 65% cationic lipid. In some embodiments, the ratio oflipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1,10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticleformulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the lipid nanoparticleformulations. As a non-limiting example, lipid nanoparticle formulationsmay contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5%to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, an respiratory virus RNA (e.g. mRNA) vaccineformulation is a nanoparticle that comprises at least one lipid. Thelipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA,98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids and amino alcohol lipids. In some embodiments, thelipid may be a cationic lipid such as, but not limited to, DLin-DMA,DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.The amino alcohol cationic lipid may be the lipids described in and/ormade by the methods described in U.S. Patent Publication No.US20130150625, herein incorporated by reference in its entirety. As anon-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, a lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, a lipid nanoparticle formulation includes 25% to75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

In some embodiments, a lipid nanoparticle formulation includes 0.5% to15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include,without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments,the formulation includes 5% to 50% on a molar basis of the sterol (e.g.,15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. Anon-limiting example of a sterol is cholesterol. In some embodiments, alipid nanoparticle formulation includes 0.5% to 20% on a molar basis ofthe PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%,1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprisesa PEG molecule of an average molecular weight of less than 2,000, forexample around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limitingexamples of PEG-modified lipids include PEG-distearoyl glycerol(PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA(further discussed in Reyes et al. J. Controlled Release, 107, 276-287(2005) the contents of which are herein incorporated by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations include 25-75% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 35-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 45-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 60% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of theneutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modifiedlipid, and 0.5% of the targeting lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 40% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of theneutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.2% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of theneutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the contents of which are herein incorporated by reference intheir entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and3.5% of the PEG or PEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in molar ratios of 20-70% cationiclipid:5-45% neutral lipid:20-55% cholesterol: 0.5-15% PEG-modifiedlipid. In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in a molar ratio of 20-60% cationiclipid:5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modifiedlipid.

In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationiclipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g.,PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid,e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Non-limiting examples of lipid nanoparticle compositions and methods ofmaking them are described, for example, in Semple et al. (2010) Nat.Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed.,51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578(the contents of each of which are incorporated herein by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations may comprise acationic lipid, a PEG lipid and a structural lipid and optionallycomprise a non-cationic lipid. As a non-limiting example, a lipidnanoparticle may comprise 40-60% of cationic lipid, 5-15% of anon-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structurallipid. As another non-limiting example, the lipid nanoparticle maycomprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and38.5% structural lipid. As yet another non-limiting example, a lipidnanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid,2.5% PEG lipid and 32.5% structural lipid. In some embodiments, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2%of a PEG lipid and 30-50% of a structural lipid. As another non-limitingexample, the lipid nanoparticle may comprise 50% cationic lipid, 10%non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle may comprise 55%cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-KC2-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of thestructural lipid cholesterol. As yet another non-limiting example, thelipid nanoparticle comprise 55% of the cationic lipid L319, 10% of thenon-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of thestructural lipid cholesterol.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in a vaccinecomposition may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the composition is to be administered. For example, thecomposition may comprise between 0.1% and 99% (w/w) of the activeingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, atleast 80% (w/w) active ingredient.

In some embodiments, the respiratory virus RNA (e.g. mRNA) vaccinecomposition may comprise the polynucleotide described herein, formulatedin a lipid nanoparticle comprising MC3, Cholesterol, DSPC andPEG2000-DMG, the buffer trisodium citrate, sucrose and water forinjection. As a non-limiting example, the composition comprises: 2.0mg/mL of drug substance (e.g., polynucleotides encoding H10N8 hMPV),21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7mg/mL of PEG2000-DMG, 5.16 mg/mL of trisodium citrate, 71 mg/mL ofsucrose and 1.0 mL of water for injection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

Liposomes, Lipoplexes, and Lipid Nanoparticles

The RNA (e.g., mRNA) vaccines of the disclosure can be formulated usingone or more liposomes, lipoplexes, or lipid nanoparticles. In someembodiments, pharmaceutical compositions of RNA (e.g., mRNA) vaccinesinclude liposomes. Liposomes are artificially-prepared vesicles whichmay primarily be composed of a lipid bilayer and may be used as adelivery vehicle for the administration of nutrients and pharmaceuticalformulations. Liposomes can be of different sizes such as, but notlimited to, a multilamellar vesicle (MLV) which may be hundreds ofnanometers in diameter and may contain a series of concentric bilayersseparated by narrow aqueous compartments, a small unicellular vesicle(SUV) which may be smaller than 50 nm in diameter, and a largeunilamellar vesicle (LUV) which may be between 50 and 500 nm indiameter. Liposome design may include, but is not limited to, opsoninsor ligands in order to improve the attachment of liposomes to unhealthytissue or to activate events such as, but not limited to, endocytosis.Liposomes may contain a low or a high pH in order to improve thedelivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In some embodiments, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104; all of which are incorporatedherein in their entireties). The original manufacture method by Wheeleret al. was a detergent dialysis method, which was later improved byJeffs et al. and is referred to as the spontaneous vesicle formationmethod. The liposome formulations are composed of 3 to 4 lipidcomponents in addition to the polynucleotide. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations may contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations may comprise from about 25.0%cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol toabout 45.0% cholesterol, from about 35.0% cholesterol to about 50.0%cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. In some embodiments, formulations may comprise a percentageof cholesterol selected from the group consisting of 28.5%, 31.5%,33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments,formulations may comprise from about 5.0% to about 10.0% DSPC and/orfrom about 7.0% to about 15.0% DSPC.

In some embodiments, the RNA (e.g., mRNA) vaccine pharmaceuticalcompositions may be formulated in liposomes such as, but not limited to,DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (MarinaBiotech, Bothell, Wash.), neutral DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNAdelivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 20065(12)1708-1713); herein incorporated by reference in its entirety) andhyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In some embodiments, the cationic lipid may be a low molecular weightcationic lipid such as those described in U.S. Patent Application No.20130090372, the contents of which are herein incorporated by referencein their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid vesicle, which may have crosslinks between functionalized lipidbilayers.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid-polycation complex. The formation of the lipid-polycationcomplex may be accomplished by methods known in the art and/or asdescribed in U.S. Pub. No. 20120178702, herein incorporated by referencein its entirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In some embodiments, theRNA (e.g., mRNA) vaccines may be formulated in a lipid-polycationcomplex, which may further include a non-cationic lipid such as, but notlimited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina lipid nanoparticle.

In some embodiments, the RNA (e.g., mRNA) vaccine formulation comprisingthe polynucleotide is a nanoparticle which may comprise at least onelipid. The lipid may be selected from, but is not limited to, DLin-DMA,DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA,PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In anotheraspect, the lipid may be a cationic lipid such as, but not limited to,DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and aminoalcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in U.S. PatentPublication No. US20130150625, herein incorporated by reference in itsentirety. As a non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, the lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, the formulation includes from about 25% to about75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In some embodiments, the formulation includes from about 0.5% to about15% on a molar basis of the neutral lipid e.g., from about 3 to about12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% ona molar basis. Examples of neutral lipids include, but are not limitedto, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulationincludes from about 5% to about 50% on a molar basis of the sterol(e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about38.5%, about 35%, or about 31% on a molar basis. An exemplary sterol ischolesterol. In some embodiments, the formulation includes from about0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid(e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis. In someembodiments, the PEG or PEG modified lipid comprises a PEG molecule ofan average molecular weight of 2,000 Da. In other embodiments, the PEGor PEG modified lipid comprises a PEG molecule of an average molecularweight of less than 2,000, for example around 1,500 Da, around 1,000 Da,or around 500 Da. Examples of PEG-modified lipids include, but are notlimited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein asPEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J.Controlled Release, 107, 276-287 (2005) the contents of which are hereinincorporated by reference in their entirety)

In some embodiments, the formulations of the present disclosure include25-75% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure include35-65% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure include45-65% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 60% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of the neutral lipid, about 31% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 38.5% of the sterol, and about 1.5% of the PEGor PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 50% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofthe neutral lipid, about 35% of the sterol, about 4.5% or about 5% ofthe PEG or PEG-modified lipid, and about 0.5% of the targeting lipid ona molar basis.

In some embodiments, the formulations of the present disclosure includeabout 40% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% ofthe neutral lipid, about 40% of the sterol, and about 5% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 57.2% of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1%of the neutral lipid, about 34.3% of the sterol, and about 1.4% of thePEG or PEG-modified lipid on a molar basis.

In some embodiments, the formulations of the present disclosure includeabout 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA(PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release,107, 276-287 (2005), the contents of which are herein incorporated byreference in their entirety), about 7.5% of the neutral lipid, about31.5% of the sterol, and about 3.5% of the PEG or PEG-modified lipid ona molar basis.

In some embodiments, lipid nanoparticle formulation consists essentiallyof a lipid mixture in molar ratios of about 20-70% cationic lipid:5-45%neutral lipid:20-55% cholesterol: 0.5-15% PEG-modified lipid; morepreferably in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% cholesterol: 0.5-15% PEG-modified lipid.

In some embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Examples of lipid nanoparticle compositions and methods of making sameare described, for example, in Semple et al. (2010) Nat. Biotechnol.28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (thecontents of each of which are incorporated herein by reference in theirentirety).

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a PEG lipid and a structural lipidand optionally comprise a non-cationic lipid. As a non-limiting example,the lipid nanoparticle may comprise about 40-60% of cationic lipid,about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about30-50% of a structural lipid. As another non-limiting example, the lipidnanoparticle may comprise about 50% cationic lipid, about 10%non-cationic lipid, about 1.5% PEG lipid and about 38.5% structurallipid. As yet another non-limiting example, the lipid nanoparticle maycomprise about 55% cationic lipid, about 10% non-cationic lipid, about2.5% PEG lipid and about 32.5% structural lipid. In some embodiments,the cationic lipid may be any cationic lipid described herein such as,but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise about 40-60% of cationic lipid, about 5-15% of a non-cationiclipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.As another non-limiting example, the lipid nanoparticle may compriseabout 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEGlipid and about 38.5% structural lipid. As yet another non-limitingexample, the lipid nanoparticle may comprise about 55% cationic lipid,about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise about 50% of the cationic lipid DLin-KC2-DMA,about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipidPEG-DOMG and about 38.5% of the structural lipid cholesterol. As anon-limiting example, the lipid nanoparticle comprise about 50% of thecationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC,about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structurallipid cholesterol. As a non-limiting example, the lipid nanoparticlecomprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of thenon-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about38.5% of the structural lipid cholesterol. As yet another non-limitingexample, the lipid nanoparticle comprise about 55% of the cationic lipidL319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEGlipid PEG-DMG and about 32.5% of the structural lipid cholesterol.

As a non-limiting example, the cationic lipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8 amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1 amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-R1S,2R)-2-octylcyclopropyllpentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)-N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-R1S,25)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-1[8-(2-oc1ylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the LNP formulations of the RNA (e.g., mRNA)vaccines may contain PEG-c-DOMG at 3% lipid molar ratio. In someembodiments, the LNP formulations of the RNA (e.g., mRNA) vaccines maycontain PEG-c-DOMG at 1.5% lipid molar ratio.

In some embodiments, the pharmaceutical compositions of the RNA (e.g.,mRNA) vaccines may include at least one of the PEGylated lipidsdescribed in International Publication No. WO2012099755, the contents ofwhich are herein incorporated by reference in their entirety.

In some embodiments, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In some embodiments, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In some embodiments, the LNP formulation may contain PEG-DMG2000, a cationic lipid known in the art, DSPC and cholesterol. As anon-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral deliveryof self-amplifying RNA (e.g., mRNA) vaccines, PNAS 2012; PMID: 22908294,the contents of each of which are herein incorporated by reference intheir entirety).

The lipid nanoparticles described herein may be made in a sterileenvironment.

In some embodiments, the LNP formulation may be formulated in ananoparticle such as a nucleic acid-lipid particle. As a non-limitingexample, the lipid particle may comprise one or more active agents ortherapeutic agents; one or more cationic lipids comprising from about 50mol % to about 85 mol % of the total lipid present in the particle; oneor more non-cationic lipids comprising from about 13 mol % to about 49.5mol % of the total lipid present in the particle; and one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 2 mol % of the total lipid present in theparticle.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. As a non-limitingexample, the phosphate conjugates may include a compound of any one ofthe formulas described in International Application No. WO2013033438,the contents of which are herein incorporated by reference in itsentirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water soluble conjugate. The polymerconjugate may have a structure as described in U.S. Patent ApplicationNo. 20130059360, the contents of which are herein incorporated byreference in its entirety. In some embodiments, polymer conjugates withthe polynucleotides of the present disclosure may be made using themethods and/or segmented polymeric reagents described in U.S. PatentApplication No. 20130072709, the contents of which are hereinincorporated by reference in its entirety. In some embodiments, thepolymer conjugate may have pendant side groups comprising ring moietiessuch as, but not limited to, the polymer conjugates described in U.S.Patent Publication No. US20130196948, the contents which are hereinincorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present disclosure in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate may be a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al. (Science 2013 339, 971-975),herein incorporated by reference in its entirety). As shown by Rodriguezet al., the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles. In anotheraspect, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure are formulated in nanoparticles which comprise a conjugate toenhance the delivery of the nanoparticles of the present disclosure in asubject. The conjugate may be the CD47 membrane or the conjugate may bederived from the CD47 membrane protein, such as the “self” peptidedescribed previously. In some embodiments, the nanoparticle may comprisePEG and a conjugate of CD47 or a derivative thereof. In someembodiments, the nanoparticle may comprise both the “self” peptidedescribed above and the membrane protein CD47.

In some embodiments, a “self” peptide and/or CD47 protein may beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the RNA (e.g., mRNA) vaccines of the present disclosure.

In some embodiments, RNA (e.g., mRNA) vaccine pharmaceuticalcompositions comprising the polynucleotides of the present disclosureand a conjugate that may have a degradable linkage. Non-limitingexamples of conjugates include an aromatic moiety comprising anionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.As a non-limiting example, pharmaceutical compositions comprising aconjugate with a degradable linkage and methods for delivering suchpharmaceutical compositions are described in U.S. Patent Publication No.US20130184443, the contents of which are herein incorporated byreference in their entirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine. As anon-limiting example, the carbohydrate carrier may include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012109121; the contents of which are hereinincorporated by reference in their entirety).

Nanoparticle formulations of the present disclosure may be coated with asurfactant or polymer in order to improve the delivery of the particle.In some embodiments, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,RNA (e.g., mRNA) vaccines within the central nervous system. As anon-limiting example nanoparticles comprising a hydrophilic coating andmethods of making such nanoparticles are described in U.S. PatentPublication No. US20130183244, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the lipid nanoparticles of the present disclosuremay be hydrophilic polymer particles. Non-limiting examples ofhydrophilic polymer particles and methods of making hydrophilic polymerparticles are described in U.S. Patent Publication No. US20130210991,the contents of which are herein incorporated by reference in theirentirety.

In some embodiments, the lipid nanoparticles of the present disclosuremay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In some embodiments, the internal ester linkage may be located on eitherside of the saturated carbon.

In some embodiments, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a polynucleotidedescribed herein. In some embodiments, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosa tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670 or International PatentPublication No. WO2013110028, the contents of each of which are hereinincorporated by reference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Patent PublicationNo. WO2013116804, the contents of which are herein incorporated byreference in their entirety. The polymeric material may additionally beirradiated. As a non-limiting example, the polymeric material may begamma irradiated (see e.g., International App. No. WO201282165, hereinincorporated by reference in its entirety). Non-limiting examples ofspecific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S.Publication 20120121718 and U.S. Publication 20100003337 and U.S. Pat.No. 8,263,665, the contents of each of which is herein incorporated byreference in their entirety). The co-polymer may be a polymer that isgenerally regarded as safe (GRAS) and the formation of the lipidnanoparticle may be in such a way that no new chemical entities arecreated. For example, the lipid nanoparticle may comprise poloxamerscoating PLGA nanoparticles without forming new chemical entities whichare still able to rapidly penetrate human mucus (Yang et al. Angew.Chem. Int. Ed. 2011 50:2597-2600; the contents of which are hereinincorporated by reference in their entirety). A non-limiting scalablemethod to produce nanoparticles which can penetrate human mucus isdescribed by Xu et al. (see, e.g., J Control Release 2013,170(2):279-86; the contents of which are herein incorporated byreference in their entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecylammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase. The surface altering agent may be embedded orenmeshed in the particle's surface or disposed (e.g., by coating,adsorption, covalent linkage, or other process) on the surface of thelipid nanoparticle. (see e.g., U.S. Publication 20100215580 and U.S.Publication 20080166414 and US20130164343; the contents of each of whichare herein incorporated by reference in their entirety).

In some embodiments, the mucus penetrating lipid nanoparticles maycomprise at least one polynucleotide described herein. Thepolynucleotide may be encapsulated in the lipid nanoparticle and/ordisposed on the surface of the particle. The polynucleotide may becovalently coupled to the lipid nanoparticle. Formulations of mucuspenetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion, which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In some embodiments, the mucus penetrating lipid nanoparticles may be ahypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation may be hypotonice for the epithelium to whichit is being delivered. Non-limiting examples of hypotonic formulationsmay be found in International Patent Publication No. WO2013110028, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, in order to enhance the delivery through themucosal barrier the RNA (e.g., mRNA) vaccine formulation may comprise orbe a hypotonic solution. Hypotonic solutions were found to increase therate at which mucoinert particles such as, but not limited to,mucus-penetrating particles, were able to reach the vaginal epithelialsurface (see e.g., Ensign et al. Biomaterials 2013 34(28):6922-9, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as alipoplex, such as, without limitation, the ATUPLEX™ system, the DACCsystem, the DBTC system and other siRNA-lipoplex technology from SilenceTherapeutics (London, United Kingdom), STEMFECT™ from STEMGENT®(Cambridge, Mass.), and polyethylenimine (PEI) or protamine-basedtargeted and non-targeted delivery of nucleic acids acids (Aleku et al.Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin PharmacolTher 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santelet al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol.Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother.2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294;Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song et al., NatureBiotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 20076; 104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132, thecontents of each of which are incorporated herein by reference in theirentirety).

In some embodiments, such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, thecontents of each of which are incorporated herein by reference in theirentirety). One example of passive targeting of formulations to livercells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipidnanoparticle formulations, which have been shown to bind toapolipoprotein E and promote binding and uptake of these formulationsinto hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364, thecontents of which are incorporated herein by reference in theirentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin DrugDeliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364;Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al.,Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release.20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kimet al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther.2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer etal., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133, the contents of each of which are incorporated herein byreference in their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as asolid lipid nanoparticle. A solid lipid nanoparticle (SLN) may bespherical with an average diameter between 10 to 1000 nm. SLN possess asolid lipid core matrix that can solubilize lipophilic molecules and maybe stabilized with surfactants and/or emulsifiers. In some embodiments,the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle(see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents ofwhich are herein incorporated by reference in their entirety). As anon-limiting example, the SLN may be the SLN described in InternationalPatent Publication No. WO2013105101, the contents of which are hereinincorporated by reference in their entirety. As another non-limitingexample, the SLN may be made by the methods or processes described inInternational Patent Publication No. WO2013105101, the contents of whichare herein incorporated by reference in their entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides directed protein production as theseformulations may be able to increase cell transfection by the RNA (e.g.,mRNA) vaccine; and/or increase the translation of encoded protein. Onesuch example involves the use of lipid encapsulation to enable theeffective systemic delivery of polyplex plasmid DNA (Heyes et al., MolTher. 2007 15: 713-720; the contents of which are incorporated herein byreference in their entirety). The liposomes, lipoplexes, or lipidnanoparticles may also be used to increase the stability of thepolynucleotide.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure can be formulated for controlled release and/or targeteddelivery. As used herein, “controlled release” refers to apharmaceutical composition or compound release profile that conforms toa particular pattern of release to effect a therapeutic outcome. In someembodiments, the RNA (e.g., mRNA) vaccines may be encapsulated into adelivery agent described herein and/or known in the art for controlledrelease and/or targeted delivery. As used herein, the term “encapsulate”means to enclose, surround or encase. As it relates to the formulationof the compounds of the disclosure, encapsulation may be substantial,complete or partial. The term “substantially encapsulated” means that atleast greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.9 or greater than 99.999% of the pharmaceutical composition orcompound of the disclosure may be enclosed, surrounded or encased withinthe delivery agent. “Partially encapsulation” means that less than 10,10, 20, 30, 40 50 or less of the pharmaceutical composition or compoundof the disclosure may be enclosed, surrounded or encased within thedelivery agent. Advantageously, encapsulation may be determined bymeasuring the escape or the activity of the pharmaceutical compositionor compound of the disclosure using fluorescence and/or electronmicrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80,85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thepharmaceutical composition or compound of the disclosure areencapsulated in the delivery agent.

In some embodiments, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and

WO2012131106, the contents of each of which are incorporated herein byreference in their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulatedinto a lipid nanoparticle or a rapidly eliminated lipid nanoparticle andthe lipid nanoparticles or a rapidly eliminated lipid nanoparticle maythen be encapsulated into a polymer, hydrogel and/or surgical sealantdescribed herein and/or known in the art. As a non-limiting example, thepolymer, hydrogel or surgical sealant may be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

In some embodiments, the lipid nanoparticle may be encapsulated into anypolymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In some embodiments, the RNA (e.g., mRNA) vaccine formulation forcontrolled release and/or targeted delivery may also include at leastone controlled release coating. Controlled release coatings include, butare not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetatecopolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose,hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGITRS® and cellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releaseand/or targeted delivery formulation may comprise at least onedegradable polyester which may contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In some embodiments, the degradable polyestersmay include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releaseand/or targeted delivery formulation comprising at least onepolynucleotide may comprise at least one PEG and/or PEG related polymerderivatives as described in U.S. Pat. No. 8,404,222, the contents ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releasedelivery formulation comprising at least one polynucleotide may be thecontrolled release polymer system described in US20130130348, thecontents of which are incorporated herein by reference in theirentirety.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be encapsulated in a therapeutic nanoparticle, referredto herein as “therapeutic nanoparticle RNA (e.g., mRNA) vaccines.”Therapeutic nanoparticles may be formulated by methods described hereinand known in the art such as, but not limited to, International Pub Nos.WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923,U.S. Publication Nos. US20110262491, US20100104645, US20100087337,US20100068285, US20110274759, US20100068286, US20120288541,US20130123351 and US20130230567 and U.S. Pat. Nos. 8,206,747, 8,293,276,8,318,208 and 8,318,211; the contents of each of which are hereinincorporated by reference in their entirety. In some embodiments,therapeutic polymer nanoparticles may be identified by the methodsdescribed in US Pub No. US20120140790, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA)vaccine may be formulated for sustained release. As used herein,“sustained release” refers to a pharmaceutical composition or compoundthat conforms to a release rate over a specific period of time. Theperiod of time may include, but is not limited to, hours, days, weeks,months and years. As a non-limiting example, the sustained releasenanoparticle may comprise a polymer and a therapeutic agent such as, butnot limited to, the polynucleotides of the present disclosure (seeInternational Pub No. 2010075072 and US Pub No. US20100216804,US20110217377 and US20120201859, the contents of each of which areincorporated herein by reference in their entirety). In anothernon-limiting example, the sustained release formulation may compriseagents which permit persistent bioavailability such as, but not limitedto, crystals, macromolecular gels and/or particulate suspensions (seeU.S. Patent Publication No US20130150295, the contents of each of whichare incorporated herein by reference in their entirety).

In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA)vaccines may be formulated to be target specific. As a non-limitingexample, the therapeutic nanoparticles may include a corticosteroid (seeInternational Pub. No. WO2011084518, the contents of which areincorporated herein by reference in their entirety). As a non-limitingexample, the therapeutic nanoparticles may be formulated innanoparticles described in International Pub No. WO2008121949,WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426,US20120004293 and US20100104655, the contents of each of which areincorporated herein by reference in their entirety.

In some embodiments, the nanoparticles of the present disclosure maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In some embodiments, the therapeutic nanoparticle comprises a diblockcopolymer. In some embodiments, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In yet anotherembodiment, the diblock copolymer may be a high-X diblock copolymer suchas those described in International Patent Publication No. WO2013120052,the contents of which are incorporated herein by reference in theirentirety.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see U.S. Publication No. US20120004293 andU.S. Pat. No. 8,236,330, each of which is herein incorporated byreference in their entirety). In another non-limiting example, thetherapeutic nanoparticle is a stealth nanoparticle comprising a diblockcopolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968and International Publication No. WO2012166923, the contents of each ofwhich are herein incorporated by reference in their entirety). In yetanother non-limiting example, the therapeutic nanoparticle is a stealthnanoparticle or a target-specific stealth nanoparticle as described inU.S. Patent Publication No. US20130172406, the contents of which areherein incorporated by reference in their entirety.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and U.S. Patent Pub. No. US20130195987, the contents of each of whichare herein incorporated by reference in their entirety).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee etal. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000;as a controlled gene delivery system in Li et al. Controlled GeneDelivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253, the contents of each of which are herein incorporated byreference in their entirety). The RNA (e.g., mRNA) vaccines of thepresent disclosure may be formulated in lipid nanoparticles comprisingthe PEG-PLGA-PEG block copolymer.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and U.S. Patent Pub. No. US20130195987, the contents of each of whichare herein incorporated by reference in their entirety).

In some embodiments, the block copolymers described herein may beincluded in a polyion complex comprising a non-polymeric micelle and theblock copolymer. (see e.g., U.S. Publication No. 20120076836, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In some embodiments, the therapeutic nanoparticles may comprise at leastone poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer may have a structure such as those described inInternational Application No. WO2013032829 or U.S. Patent Publication NoUS20130121954, the contents of each of which are herein incorporated byreference in their entirety. In some embodiments, the poly(vinyl ester)polymers may be conjugated to the polynucleotides described herein.

In some embodiments, the therapeutic nanoparticle may comprise at leastone diblock copolymer. The diblock copolymer may be, but it not limitedto, a poly(lactic) acid-poly(ethylene)glycol copolymer (see, e.g.,International Patent Publication No. WO2013044219, the contents of whichare herein incorporated by reference in their entirety). As anon-limiting example, the therapeutic nanoparticle may be used to treatcancer (see International publication No. WO2013044219, the contents ofwhich are herein incorporated by reference in their entirety).

In some embodiments, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In some embodiments, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(see, e.g., U.S. Pat. No. 8,287,849, the contents of which are hereinincorporated by reference in their entirety) and combinations thereof.

In some embodiments, the nanoparticles described herein may comprise anamine cationic lipid such as those described in International PatentApplication No. WO2013059496, the contents of which are hereinincorporated by reference in their entirety. In some embodiments, thecationic lipids may have an amino-amine or an amino-amide moiety.

In some embodiments, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In some embodiments, the degradable polyestersmay include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent, which mayenhance a Th1-based response of the immune system (see International PubNo. WO2010123569 and U.S. Publication No. US20110223201, the contents ofeach of which are herein incorporated by reference in their entirety).

In some embodiments, the synthetic nanocarriers may be formulated fortargeted release. In some embodiments, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle may be formulated to release the RNA (e.g., mRNA) vaccinesafter 24 hours and/or at a pH of 4.5 (see International Publication Nos.WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 andUS20110027217, each of which is herein incorporated by reference intheir entireties).

In some embodiments, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release may be formulated by methods known in the art,described herein and/or as described in International Pub No.WO2010138192 and US Pub No. 20100303850, each of which is hereinincorporated by reference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated forcontrolled and/or sustained release wherein the formulation comprises atleast one polymer that is a crystalline side chain (CYSC) polymer. CYSCpolymers are described in U.S. Pat. No. 8,399,007, herein incorporatedby reference in its entirety.

In some embodiments, the synthetic nanocarrier may be formulated for useas a vaccine. In some embodiments, the synthetic nanocarrier mayencapsulate at least one polynucleotide which encode at least oneantigen. As a non-limiting example, the synthetic nanocarrier mayinclude at least one antigen and an excipient for a vaccine dosage form(see International Publication No. WO2011150264 and U.S. Publication No.US20110293723, the contents of each of which are herein incorporated byreference in their entirety). As another non-limiting example, a vaccinedosage form may include at least two synthetic nanocarriers with thesame or different antigens and an excipient (see InternationalPublication No. WO2011150249 and U.S. Publication No. US20110293701, thecontents of each of which are herein incorporated by reference in theirentirety). The vaccine dosage form may be selected by methods describedherein, known in the art and/or described in International PublicationNo. WO2011150258 and U.S. Publication No. US20120027806, the contents ofeach of which are herein incorporated by reference in their entirety).

In some embodiments, the synthetic nanocarrier may comprise at least onepolynucleotide which encodes at least one adjuvant. As non-limitingexample, the adjuvant may comprise dimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium(see, e.g., U.S. Pat. No. 8,241,610, the content of which is hereinincorporated by reference in its entirety). In some embodiments, thesynthetic nanocarrier may comprise at least one polynucleotide and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Publication No. WO2011150240 and U.S. Publication No.US20110293700, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, the synthetic nanocarrier may encapsulate at leastone polynucleotide that encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier may include,but is not limited to, any of the nanocarriers described inInternational Publication No. WO2012024621, WO201202629, WO2012024632and U.S. Publication No. US20120064110, US20120058153 and US20120058154,the contents of each of which are herein incorporated by reference intheir entirety.

In some embodiments, the synthetic nanocarrier may be coupled to apolynucleotide which may be able to trigger a humoral and/or cytotoxic Tlymphocyte (CTL) response (see, e.g., International Publication No.WO2013019669, the contents of which are herein incorporated by referencein their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccine may be encapsulatedin, linked to and/or associated with zwitterionic lipids. Non-limitingexamples of zwitterionic lipids and methods of using zwitterionic lipidsare described in U.S. Patent Publication No. US20130216607, the contentsof which are herein incorporated by reference in their entirety. In someaspects, the zwitterionic lipids may be used in the liposomes and lipidnanoparticles described herein.

In some embodiments, the RNA (e.g., mRNA) vaccine may be formulated incolloid nanocarriers as described in U.S. Patent Publication No.US20130197100, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Publication No. 20120282343, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832, the contentsof which are herein incorporated by reference in their entirety.Activity and/or safety (as measured by examining one or more of ALT/AST,white blood cell count and cytokine induction, for example) of LNPadministration may be improved by incorporation of such lipids. LNPscomprising KL52 may be administered intravenously and/or in one or moredoses. In some embodiments, administration of LNPs comprising KL52results in equal or improved mRNA and/or protein expression as comparedto LNPs comprising MC3.

In some embodiments, RNA (e.g., mRNA) vaccine may be delivered usingsmaller LNPs. Such particles may comprise a diameter from below 0.1 umup to 100 nm such as, but not limited to, less than 0.1 um, less than1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20um, less than 25 um, less than 30 um, less than 35 um, less than 40 um,less than 50 um, less than 55 um, less than 60 um, less than 65 um, lessthan 70 um, less than 75 um, less than 80 um, less than 85 um, less than90 um, less than 95 um, less than 100 um, less than 125 um, less than150 um, less than 175 um, less than 200 um, less than 225 um, less than250 um, less than 275 um, less than 300 um, less than 325 um, less than350 um, less than 375 um, less than 400 um, less than 425 um, less than450 um, less than 475 um, less than 500 um, less than 525 um, less than550 um, less than 575 um, less than 600 um, less than 625 um, less than650 um, less than 675 um, less than 700 um, less than 725 um, less than750 um, less than 775 um, less than 800 um, less than 825 um, less than850 um, less than 875 um, less than 900 um, less than 925 um, less than950 um, less than 975 um, or less than 1000 um.

In some embodiments, RNA (e.g., mRNA) vaccines may be delivered usingsmaller LNPs, which may comprise a diameter from about 1 nm to about 100nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, fromabout 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm toabout 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, fromabout 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm toabout 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nm,from about 20 to about 50 nm, from about 30 to about 50 nm, from about40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, fromabout 30 to about 70 nm, from about 40 to about 70 nm, from about 50 toabout 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm,from about 30 to about 80 nm, from about 40 to about 80 nm, from about50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, fromabout 50 to about 90 nm, from about 60 to about 90 nm and/or from about70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Examples of microfluidic mixers may include, butare not limited to, a slit interdigital micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51, the contents of each of which are herein incorporatedby reference in their entirety). In some embodiments, methods of LNPgeneration comprising SHM, further comprise the mixing of at least twoinput streams wherein mixing occurs by microstructure-induced chaoticadvection (MICA). According to this method, fluid streams flow throughchannels present in a herringbone pattern causing rotational flow andfolding the fluids around each other. This method may also comprise asurface for fluid mixing wherein the surface changes orientations duringfluid cycling. Methods of generating LNPs using SHM include thosedisclosed in U.S. Application Publication Nos. 2004/0262223 and2012/0276209, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccine of the presentdisclosure may be formulated in lipid nanoparticles created using amicromixer such as, but not limited to, a Slit InterdigitalMicrostructured Mixer (SIMM-V2) or a Standard Slit Interdigital MicroMixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from theInstitut für Mikrotechnik Mainz GmbH, Mainz Germany).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles created usingmicrofluidic technology (see, e.g., Whitesides, George M. The Originsand the Future of Microfluidics. Nature, 2006 442: 368-373; and Abrahamet al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; eachof which is herein incorporated by reference in its entirety). As anon-limiting example, controlled microfluidic formulation includes apassive method for mixing streams of steady pressure-driven flows inmicro channels at a low Reynolds number (see, e.g., Abraham et al.Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, thecontents of which are herein incorporated by reference in theirentirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles created using amicromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure maybe formulated for delivery using the drug encapsulating microspheresdescribed in International Patent Publication No. WO2013063468 or U.S.Pat. No. 8,440,614, the contents of each of which are hereinincorporated by reference in their entirety. The microspheres maycomprise a compound of the formula (I), (II), (III), (IV), (V) or (VI)as described in International Patent Publication No. WO2013063468, thecontents of which are herein incorporated by reference in theirentirety. In some embodiments, the amino acid, peptide, polypeptide,lipids (APPL) are useful in delivering the RNA (e.g., mRNA) vaccines ofthe disclosure to cells (see International Patent Publication No.WO2013063468, the contents of which are herein incorporated by referencein their entirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure maybe formulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In some embodiments, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In some embodiments, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In some embodiments, the lipid nanoparticle may be a limit size lipidnanoparticle described in International Patent Publication No.WO2013059922, the contents of which are herein incorporated by referencein their entirety. The limit size lipid nanoparticle may comprise alipid bilayer surrounding an aqueous core or a hydrophobic core; wherethe lipid bilayer may comprise a phospholipid such as, but not limitedto, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, aceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, acerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments,the limit size lipid nanoparticle may comprise a polyethyleneglycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEGand DSPE-PEG.

In some embodiments, the RNA (e.g., mRNA) vaccines may be delivered,localized and/or concentrated in a specific location using the deliverymethods described in International Patent Publication No. WO2013063530,the contents of which are herein incorporated by reference in theirentirety. As a non-limiting example, a subject may be administered anempty polymeric particle prior to, simultaneously with or afterdelivering the RNA (e.g., mRNA) vaccines to the subject. The emptypolymeric particle undergoes a change in volume once in contact with thesubject and becomes lodged, embedded, immobilized or entrapped at aspecific location in the subject.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated inan active substance release system (see, e.g., U.S. Patent PublicationNo. US20130102545, the contents of which are herein incorporated byreference in their entirety). The active substance release system maycomprise 1) at least one nanoparticle bonded to an oligonucleotideinhibitor strand which is hybridized with a catalytically active nucleicacid and 2) a compound bonded to at least one substrate molecule bondedto a therapeutically active substance (e.g., polynucleotides describedherein), where the therapeutically active substance is released by thecleavage of the substrate molecule by the catalytically active nucleicacid.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina nanoparticle comprising an inner core comprising a non-cellularmaterial and an outer surface comprising a cellular membrane. Thecellular membrane may be derived from a cell or a membrane derived froma virus. As a non-limiting example, the nanoparticle may be made by themethods described in International Patent Publication No. WO2013052167,the contents of which are herein incorporated by reference in theirentirety. As another non-limiting example, the nanoparticle described inInternational Patent Publication No. WO2013052167, the contents of whichare herein incorporated by reference in their entirety, may be used todeliver the RNA (e.g., mRNA) vaccines described herein.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated inporous nanoparticle-supported lipid bilayers (protocells). Protocellsare described in International Patent Publication No. WO2013056132, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, the RNA (e.g., mRNA) vaccines described herein maybe formulated in polymeric nanoparticles as described in or made by themethods described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and EuropeanPatent No. EP2073848B1, the contents of each of which are hereinincorporated by reference in their entirety. As a non-limiting example,the polymeric nanoparticle may have a high glass transition temperaturesuch as the nanoparticles described in or nanoparticles made by themethods described in U.S. Pat. No. 8,518,963, the contents of which areherein incorporated by reference in their entirety. As anothernon-limiting example, the polymer nanoparticle for oral and parenteralformulations may be made by the methods described in European Patent No.EP2073848B1, the contents of which are herein incorporated by referencein their entirety.

In some embodiments, the RNA (e.g., mRNA) vaccines described herein maybe formulated in nanoparticles used in imaging. The nanoparticles may beliposome nanoparticles such as those described in U.S. PatentPublication No US20130129636, herein incorporated by reference in itsentirety. As a non-limiting example, the liposome may comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see, e.g.,U.S. Patent Publication No US20130129636, the contents of which areherein incorporated by reference in their entirety).

In some embodiments, the nanoparticles which may be used in the presentdisclosure are formed by the methods described in U.S. PatentApplication No. US20130130348, the contents of which are hereinincorporated by reference in their entirety.

The nanoparticles of the present disclosure may further includenutrients such as, but not limited to, those which deficiencies can leadto health hazards from anemia to neural tube defects (see, e.g., thenanoparticles described in International Patent Publication NoWO2013072929, the contents of which are herein incorporated by referencein their entirety). As a non-limiting example, the nutrient may be ironin the form of ferrous, ferric salts or elemental iron, iodine, folicacid, vitamins or micronutrients.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in a swellable nanoparticle. The swellablenanoparticle may be, but is not limited to, those described in U.S. Pat.No. 8,440,231, the contents of which are herein incorporated byreference in their entirety. As a non-limiting embodiment, the swellablenanoparticle may be used for delivery of the RNA (e.g., mRNA) vaccinesof the present disclosure to the pulmonary system (see, e.g., U.S. Pat.No. 8,440,231, the contents of which are herein incorporated byreference in their entirety).

The RNA (e.g., mRNA) vaccines of the present disclosure may beformulated in polyanhydride nanoparticles such as, but not limited to,those described in U.S. Pat. No. 8,449,916, the contents of which areherein incorporated by reference in their entirety.

The nanoparticles and microparticles of the present disclosure may begeometrically engineered to modulate macrophage and/or the immuneresponse. In some embodiments, the geometrically engineered particlesmay have varied shapes, sizes and/or surface charges in order toincorporated the polynucleotides of the present disclosure for targeteddelivery such as, but not limited to, pulmonary delivery (see, e.g.,International Publication No WO2013082111, the contents of which areherein incorporated by reference in their entirety). Other physicalfeatures the geometrically engineering particles may have include, butare not limited to, fenestrations, angled arms, asymmetry and surfaceroughness, charge which can alter the interactions with cells andtissues. As a non-limiting example, nanoparticles of the presentdisclosure may be made by the methods described in InternationalPublication No WO2013082111, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, the nanoparticles of the present disclosure may bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013090601, the contents of which areherein incorporated by reference in their entirety. The nanoparticlesmay be inorganic nanoparticles which have a compact and zwitterionicligand in order to exhibit good water solubility. The nanoparticles mayalso have small hydrodynamic diameters (HD), stability with respect totime, pH, and salinity and a low level of non-specific protein binding.

In some embodiments the nanoparticles of the present disclosure may bedeveloped by the methods described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the nanoparticles of the present disclosure arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety. The nanoparticles of the present disclosuremay be made by the methods described in U.S. Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, the stealth or target-specific stealthnanoparticles may comprise a polymeric matrix. The polymeric matrix maycomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In some embodiments, the nanoparticle may be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure maymade by the methods described in U.S. Patent Publication No.US20130171646, the contents of which are herein incorporated byreference in their entirety. The nanoparticle may comprise a nucleicacid such as, but not limited to, polynucleotides described hereinand/or known in the art.

At least one of the nanoparticles of the present disclosure may beembedded in in the core a nanostructure or coated with a low densityporous 3-D structure or coating which is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure. Non-limiting examples of the nanostructures comprising atleast one nanoparticle are described in International Patent PublicationNo. WO2013123523, the contents of which are herein incorporated byreference in their entirety.

In some embodiments the RNA (e.g., mRNA) vaccine may be associated witha cationic or polycationic compounds, including protamine, nucleoline,spermine or spermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP²² derived or analog peptides,Pestivirus Ems, HSV, VP²² (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs), PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,Calcitonin peptide(s), Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan,Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,histones, cationic polysaccharides, for example chitosan, polybrene,cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP,DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC,DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolaminechloride, CLIP 1:rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole), etc.

In other embodiments the RNA (e.g., mRNA) vaccine is not associated witha cationic or polycationic compounds.

In some embodiments, a nanoparticle comprises compounds of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)O R, andC(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,

—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)O R, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR,

and —C(═NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆

carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a5- to 14-membered heteroaryl having one or more heteroatoms selectedfrom N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉)N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

-   -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅-20 alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected

from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q isOH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In further embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

and salts and isomers thereof.

In some embodiments, a nanoparticle comprises the following compound:

or salts and isomers thereof.

In some embodiments, the disclosure features a nanoparticle compositionincluding a lipid component comprising a compound as described herein(e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe)).

In some embodiments, the disclosure features a pharmaceuticalcomposition comprising a nanoparticle composition according to thepreceding embodiments and a pharmaceutically acceptable carrier. Forexample, the pharmaceutical composition is refrigerated or frozen forstorage and/or shipment (e.g., being stored at a temperature of 4° C. orlower, such as a temperature between about −150° C. and about 0° C. orbetween about −80° C. and about −20° C. (e.g., about −5° C., −10° C.,−15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C., −130° C. or −150° C.). For example, the pharmaceuticalcomposition is a solution that is refrigerated for storage and/orshipment at, for example, about −20° C., −30° C., −40° C., −50° C., −60°C., −70° C., or −80° C.

In some embodiments, the disclosure provides a method of delivering atherapeutic and/or prophylactic (e.g., RNA, such as mRNA) to a cell(e.g., a mammalian cell). This method includes the step of administeringto a subject (e.g., a mammal, such as a human) a nanoparticlecomposition including (i) a lipid component including a phospholipid(such as a polyunsaturated lipid), a PEG lipid, a structural lipid, anda compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(lie) and (ii) a therapeutic and/or prophylactic, in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the cell.

In some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell (e.g., a mammalian cell). The methodincludes the step of contacting the cell with a nanoparticle compositionincluding (i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)an mRNA encoding the polypeptide of interest, whereby the mRNA iscapable of being translated in the cell to produce the polypeptide.

In some embodiments, the disclosure provides a method of treating adisease or disorder in a mammal (e.g., a human) in need thereof. Themethod includes the step of administering to the mammal atherapeutically effective amount of a nanoparticle composition including(i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)a therapeutic and/or prophylactic (e.g., an mRNA). In some embodiments,the disease or disorder is characterized by dysfunctional or aberrantprotein or polypeptide activity. For example, the disease or disorder isselected from the group consisting of rare diseases, infectiousdiseases, cancer and proliferative diseases, genetic diseases (e.g.,cystic fibrosis), autoimmune diseases, diabetes, neurodegenerativediseases, cardio- and reno-vascular diseases, and metabolic diseases.

In some embodiments, the disclosure provides a method of delivering(e.g., specifically delivering) a therapeutic and/or prophylactic to amammalian organ (e.g., a liver, spleen, lung, or femur). This methodincludes the step of administering to a subject (e.g., a mammal) ananoparticle composition including (i) a lipid component including aphospholipid, a PEG lipid, a structural lipid, and a compound of Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii) atherapeutic and/or prophylactic (e.g., an mRNA), in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the target organ(e.g., a liver, spleen, lung, or femur).

In some embodiments, the disclosure features a method for the enhanceddelivery of a therapeutic and/or prophylactic (e.g., an mRNA) to atarget tissue (e.g., a liver, spleen, lung, or femur). This methodincludes administering to a subject (e.g., a mammal) a nanoparticlecomposition, the composition including (i) a lipid component including acompound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe), a phospholipid, a structural lipid, and a PEG lipid; and (ii) atherapeutic and/or prophylactic, the administering including contactingthe target tissue with the nanoparticle composition, whereby thetherapeutic and/or prophylactic is delivered to the target tissue.

In some embodiments, the disclosure features a method of loweringimmunogenicity comprising introducing the nanoparticle composition ofthe disclosure into cells, wherein the nanoparticle composition reducesthe induction of the cellular immune response of the cells to thenanoparticle composition, as compared to the induction of the cellularimmune response in cells induced by a reference composition whichcomprises a reference lipid instead of a compound of Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe). For example, the cellularimmune response is an innate immune response, an adaptive immuneresponse, or both.

The disclosure also includes methods of synthesizing a compound ofFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and methodsof making a nanoparticle composition including a lipid componentcomprising the compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe).

Modes of Vaccine Administration

Respiratory virus RNA (e.g. mRNA) vaccines may be administered by anyroute which results in a therapeutically effective outcome. Theseinclude, but are not limited, to intradermal, intramuscular, and/orsubcutaneous administration. The present disclosure provides methodscomprising administering RNA (e.g., mRNA) vaccines to a subject in needthereof. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Respiratory virusRNA (e.g., mRNA) vaccines compositions are typically formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of RNA (e.g.,mRNA) vaccine compositions may be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In some embodiments, respiratory virus RNA (e.g. mRNA) vaccinescompositions may be administered at dosage levels sufficient to deliver0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, ofsubject body weight per day, one or more times a day, per week, permonth, etc. to obtain the desired therapeutic, diagnostic, prophylactic,or imaging effect (see, e.g., the range of unit doses described inInternational Publication No WO2013078199, the contents of which areherein incorporated by reference in their entirety). The desired dosagemay be delivered three times a day, two times a day, once a day, everyother day, every third day, every week, every two weeks, every threeweeks, every four weeks, every 2 months, every three months, every 6months, etc. In some embodiments, the desired dosage may be deliveredusing multiple administrations (e.g., two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, respiratory virus RNA (e.g., mRNA) vaccines compositionsmay be administered at dosage levels sufficient to deliver 0.0005 mg/kgto 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g.,about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, respiratory virus RNA (e.g., mRNA) vaccinecompositions may be administered once or twice (or more) at dosagelevels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, respiratory virus RNA (e.g., mRNA) vaccinecompositions may be administered twice (e.g., Day 0 and Day 7, Day 0 andDay 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 andDay 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later,Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 yearslater, Day 0 and 5 years later, or Day 0 and 10 years later) at a totaldose of or at dosage levels sufficient to deliver a total dose of 0.0100mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lowerdosages and frequency of administration are encompassed by the presentdisclosure. For example, a respiratory virus RNA (e.g., mRNA) vaccinecomposition may be administered three or four times.

In some embodiments, respiratory virus RNA (e.g., mRNA) vaccinecompositions may be administered twice (e.g., Day 0 and Day 7, Day 0 andDay 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 andDay 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later,Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 yearslater, Day 0 and 5 years later, or Day 0 and 10 years later) at a totaldose of or at dosage levels sufficient to deliver a total dose of 0.010mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments, the respiratory virus RNA (e.g., mRNA) vaccine foruse in a method of vaccinating a subject is administered to the subjectas a single dosage of between 10 μg/kg and 400 μg/kg of the nucleic acidvaccine (in an effective amount to vaccinate the subject). In someembodiments the RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as a single dosageof between 10 μg and 400 μg of the nucleic acid vaccine (in an effectiveamount to vaccinate the subject). In some embodiments, a respiratoryvirus RNA (e.g., mRNA) vaccine for use in a method of vaccinating asubject is administered to the subject as a single dosage of 25-1000 μg(e.g., a single dosage of mRNA encoding hMPV, PIV3, RSV, MeV and/orBetaCoV antigen). In some embodiments, a respiratory virus RNA (e.g.,mRNA) vaccine is administered to the subject as a single dosage of 25,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950 or 1000 μg. For example, a respiratory virus RNA(e.g., mRNA) vaccine may be administered to a subject as a single doseof 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500,250-1000, or 500-1000 μg. In some embodiments, a respiratory virus RNA(e.g., mRNA) vaccine for use in a method of vaccinating a subject isadministered to the subject as two dosages, the combination of whichequals 25-1000 μg of the respiratory virus RNA (e.g., mRNA) vaccine.

A respiratory virus RNA (e.g. mRNA) vaccine pharmaceutical compositiondescribed herein can be formulated into a dosage form described herein,such as an intranasal, intratracheal, or injectable (e.g., intravenous,intraocular, intravitreal, intramuscular, intradermal, intracardiac,intraperitoneal, and subcutaneous).

Respiratory Virus RNA (e.g., mRNA) Vaccine Formulations and Methods ofUse

Some aspects of the present disclosure provide formulations of therespiratory virus RNA (e.g., mRNA) vaccine, wherein the RNA (e.g., mRNA)vaccine is formulated in an effective amount to produce an antigenspecific immune response in a subject (e.g., production of antibodiesspecific to an hMPV, PIV3, RSV, MeV and/or BetaCoV antigenicpolypeptide). “An effective amount” is a dose of an RNA (e.g., mRNA)vaccine effective to produce an antigen-specific immune response. Alsoprovided herein are methods of inducing an antigen-specific immuneresponse in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-hMPV, anti-PIV3, anti-RSV, anti-MeVand/or anti-BetaCoV antigenic polypeptide antibody titer produced in asubject administered a respiratory virus RNA (e.g., mRNA) vaccine asprovided herein. An antibody titer is a measurement of the amount ofantibodies within a subject, for example, antibodies that are specificto a particular antigen (e.g., an anti-hMPV, anti-PIV3, anti-RSV,anti-MeV and/or anti-BetaCoV antigenic polypeptide) or epitope of anantigen. Antibody titer is typically expressed as the inverse of thegreatest dilution that provides a positive result. Enzyme-linkedimmunosorbent assay (ELISA) is a common assay for determining antibodytiters, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by therespiratory virus RNA (e.g., mRNA) vaccine.

In some embodiments, an anti-antigenic polypeptide (e.g., an anti-hMPV,anti-PIV3, anti-RSV, anti-MeV and/or anti-BetaCoV antigenic polypeptide)antibody titer produced in a subject is increased by at least 1 logrelative to a control. For example, anti-antigenic polypeptide antibodytiter produced in a subject may be increased by at least 1.5, at least2, at least 2.5, or at least 3 log relative to a control. In someembodiments, the anti-antigenic polypeptide antibody titer produced inthe subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to acontrol. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased by 1-3 log relative to acontrol. For example, the anti-antigenic polypeptide antibody titerproduced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2,1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-antigenic polypeptide (e.g., an anti-hMPV,anti-PIV3, anti-RSV, anti-MeV and/or anti-BetaCoV antigenic polypeptide)antibody titer produced in a subject is increased at least 2 timesrelative to a control. For example, the anti-antigenic polypeptideantibody titer produced in a subject may be increased at least 3 times,at least 4 times, at least 5 times, at least 6 times, at least 7 times,at least 8 times, at least 9 times, or at least 10 times relative to acontrol. In some embodiments, the anti-antigenic polypeptide antibodytiter produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10times relative to a control. In some embodiments, the anti-antigenicpolypeptide antibody titer produced in a subject is increased 2-10 timesrelative to a control. For example, the anti-antigenic polypeptideantibody titer produced in a subject may be increased 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9,4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

A control, in some embodiments, is the anti-antigenic polypeptide (e.g.,an anti-hMPV, anti-PIV3, anti-RSV, anti-MeV and/or anti-BetaCoVantigenic polypeptide) antibody titer produced in a subject who has notbeen administered a respiratory virus RNA (e.g., mRNA) vaccine of thepresent disclosure. In some embodiments, a control is an anti-antigenicpolypeptide (e.g., an anti-hMPV, anti-PIV3, anti-RSV, anti-MeV and/oranti-BetaCoV antigenic polypeptide) antibody titer produced in a subjectwho has been administered a live attenuated hMPV, PIV3, RSV, MeV and/orBetaCoV vaccine. An attenuated vaccine is a vaccine produced by reducingthe virulence of a viable (live). An attenuated virus is altered in amanner that renders it harmless or less virulent relative to live,unmodified virus. In some embodiments, a control is an anti-antigenicpolypeptide (e.g., an anti-hMPV, anti-PIV3, anti-RSV, anti-MeV and/oranti-BetaCoV antigenic polypeptide) antibody titer produced in a subjectadministered inactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine. Insome embodiments, a control is an anti-antigenic polypeptide (e.g., ananti-hMPV, anti-PIV3, anti-RSV, anti-MeV and/or anti-BetaCoV antigenicpolypeptide) antibody titer produced in a subject administered arecombinant or purified hMPV, PIV3, RSV, MeV and/or BetaCoV proteinvaccine. Recombinant protein vaccines typically include protein antigensthat either have been produced in a heterologous expression system(e.g., bacteria or yeast) or purified from large amounts of thepathogenic organism. In some embodiments, a control is an anti-antigenicpolypeptide (e.g., an anti-hMPV, anti-PIV3, anti-RSV, anti-MeV and/oranti-BetaCoV antigenic polypeptide) antibody titer produced in a subjectwho has been administered an hMPV, PIV3, RSV, MeV and/or BetaCoVvirus-like particle (VLP) vaccine. For example, an hMPV VLP vaccine usedas a control may be a hMPV VLPs, comprising (or consisting of) viralmatrix (M) and fusion (F) proteins, generated by expressing viralproteins in suspension-adapted human embryonic kidney epithelial (293-F)cells (see, e.g., Cox R G et al., J Virol. 2014 June; 88(11): 6368-6379,the contents of which are herein incorporated by reference).

In some embodiments, an effective amount of a respiratory virus RNA(e.g., mRNA) vaccine is a dose that is reduced compared to the standardof care dose of a recombinant hMPV, PIV3, RSV, MeV and/or BetaCoVprotein vaccine. A “standard of care,” as provided herein, refers to amedical or psychological treatment guideline and can be general orspecific. “Standard of care” specifies appropriate treatment based onscientific evidence and collaboration between medical professionalsinvolved in the treatment of a given condition. It is the diagnostic andtreatment process that a physician/clinician should follow for a certaintype of patient, illness or clinical circumstance. A “standard of caredose,” as provided herein, refers to the dose of a recombinant orpurified hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine, or a liveattenuated or inactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine,that a physician/clinician or other medical professional wouldadminister to a subject to treat or prevent hMPV, PIV3, RSV, MeV and/orBetaCoV, or a hMPV-, PIV3-, RSV-, MeV- and/or BetaCoV-related condition,while following the standard of care guideline for treating orpreventing hMPV, PIV3, RSV, MeV and/or BetaCoV, or a hMPV-, PIV3-, RSV-,MeV- and/or BetaCoV-related condition.

In some embodiments, the anti-antigenic polypeptide (e.g., an anti-hMPV,anti-PIV3, anti-RSV, anti-MeV and/or anti-BetaCoV antigenic polypeptide)antibody titer produced in a subject administered an effective amount ofa respiratory virus RNA (e.g., mRNA) vaccine is equivalent to ananti-antigenic polypeptide (e.g., an anti-hMPV, anti-PIV3, anti-RSV,anti-MeV and/or anti-BetaCoV antigenic polypeptide) antibody titerproduced in a control subject administered a standard of care dose of arecombinant or purified hMPV, PIV3, RSV, MeV and/or BetaCoV proteinvaccine or a live attenuated or inactivated hMPV, PIV3, RSV, MeV and/orBetaCoV vaccine.

In some embodiments, an effective amount of a respiratory virus RNA(e.g., mRNA) vaccine is a dose equivalent to an at least 2-foldreduction in a standard of care dose of a recombinant or purified hMPV,PIV3, RSV, MeV and/or BetaCoV protein vaccine. For example, an effectiveamount of a respiratory virus RNA (e.g., mRNA) vaccine may be a doseequivalent to an at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold reduction in a standard of care dose of a recombinant orpurified hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine. In someembodiments, an effective amount of a respiratory virus RNA (e.g., mRNA)vaccine is a dose equivalent to an at least at least 100-fold, at least500-fold, or at least 1000-fold reduction in a standard of care dose ofa recombinant or purified hMPV, PIV3, RSV, MeV and/or BetaCoV proteinvaccine. In some embodiments, an effective amount of a respiratory virusRNA (e.g., mRNA) vaccine is a dose equivalent to a 2-, 3-, 4-, 5-, 6-,7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-fold reduction in astandard of care dose of a recombinant or purified hMPV, PIV3, RSV, MeVand/or BetaCoV protein vaccine. In some embodiments, the anti-antigenicpolypeptide antibody titer produced in a subject administered aneffective amount of a respiratory virus RNA (e.g., mRNA) vaccine isequivalent to an anti-antigenic polypeptide antibody titer produced in acontrol subject administered the standard of care dose of a recombinantor protein hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine or a liveattenuated or inactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine.In some embodiments, an effective amount of a respiratory virus RNA(e.g., mRNA) vaccine is a dose equivalent to a 2-fold to 1000-fold(e.g., 2-fold to 100-fold, 10-fold to 1000-fold) reduction in thestandard of care dose of a recombinant or purified hMPV, PIV3, RSV, MeVand/or BetaCoV protein vaccine, wherein the anti-antigenic polypeptideantibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purifiedhMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine or a live attenuatedor inactivated hMPV, PIV3, RSV, MeV and/or BetaCoV vaccine.

In some embodiments, the effective amount of a respiratory virus RNA(e.g., mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2to 800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to40-, 2 to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3to 600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4to 1000-, 4 to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to400-, 4 to 4 to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-,4 to 60-, 4 to 50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4to 8-, 4 to 7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to800-, 5 to 700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-,5 to 100-, 5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5to 30-, 5 to 20-, 5 to 10-, 5 to 9-, 5 to 8-, 5 to 7-, 5 to 6-, 6 to1000-, 6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-,6 to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-,6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-,7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7to 8-, 8 to 1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to500-, 8 to 400-, 8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8to 70-, 8 to 60-, 8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to9-, 9 to 1000-, 9 to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9to 400-, 9 to 300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-,9 to 60-, 9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-,10 to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-,10 to 300-, 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to300-, 20 to 200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to60-, 20 to 50-, 20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to800-, 30 to 700-, 30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to200-, 30 to 100-, 30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-,30 to 40-, 40 to 1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-,40 to 500-, 40 to 400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-,40 to 80-, 40 to 70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50to 800-, 50 to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50to 200-, 50 to 100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to500-, 80 to 400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to1000-, 90 to 900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to400-, 90 to 300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100to 800-, 100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to300-, 100 to 200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-,200 to 600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-,400 to 1000-, 400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-,600 to 1000-, 600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700to 900-, 700 to 800-, 800 to 1000-, 800 to 900-, or 900 to 1000-foldreduction in the standard of care dose of a recombinant hMPV, PIV3, RSV,MeV and/or BetaCoV protein vaccine. In some embodiments, theanti-antigenic polypeptide antibody titer produced in the subject isequivalent to an anti-antigenic polypeptide antibody titer produced in acontrol subject administered the standard of care dose of a recombinantor purified hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine or alive attenuated or inactivated hMPV, PIV3, RSV, MeV and/or BetaCoVvaccine. In some embodiments, the effective amount is a dose equivalentto (or equivalent to an at least) 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-,20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-,150-, 160-, 170-, 1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-,270-, 280-, 290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-,390-, 400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-,510-, 520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-,630-, 640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-,750-, 760-, 770-, 780-, 790-, 800-, 810-, 820-, 830-, 840-, 850-, 860-,870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-,990-, or 1000-fold reduction in the standard of care dose of arecombinant hMPV, PIV3, RSV, MeV and/or BetaCoV protein vaccine. In someembodiments, an anti-antigenic polypeptide antibody titer produced inthe subject is equivalent to an anti-antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a recombinant or purified hMPV, PIV3, RSV, MeV and/or BetaCoVprotein vaccine or a live attenuated or inactivated hMPV, PIV3, RSV, MeVand/or BetaCoV vaccine.

In some embodiments, the effective amount of a respiratory virus RNA(e.g., mRNA) vaccine is a total dose of 50-1000 μg. In some embodiments,the effective amount of a respiratory virus RNA (e.g., mRNA) vaccine isa total dose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400,50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900,60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90,60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400,70-300, 70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700,80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900,90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100,100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300,100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400,200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400,400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000,500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-900, 600-700,700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 μg. In someembodiments, the effective amount of a respiratory virus RNA (e.g.,mRNA) vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. Insome embodiments, the effective amount is a dose of 25-500 μgadministered to the subject a total of two times. In some embodiments,the effective amount of a respiratory virus RNA (e.g., mRNA) vaccine isa dose of 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400,50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 150-500,150-400, 150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400,250-300, 300-500, 300-400, 350-500, 350-400, 400-500 or 450-500 μgadministered to the subject a total of two times. In some embodiments,the effective amount of a respiratory virus RNA (e.g., mRNA) vaccine isa total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500μg administered to the subject a total of two times.

Examples of Additional Embodiments of the Disclosure

Additional embodiments of the present disclosure are encompassed by thefollowing numbered paragraphs:

1. A respiratory virus vaccine, comprising: at least one ribonucleicacid (RNA) polynucleotide having an open reading frame encoding at leastone, at least two, at least three, at least four or at least fiveantigenic polypeptides selected from human Metapneumovirus (hMPV)antigenic polypeptides or immunogenic fragments thereof, humanparainfluenza virus type 3 (PIV3) antigenic polypeptides or immunogenicfragments thereof, respiratory syncytial virus (RSV) antigenicpolypeptides or immunogenic fragments thereof, measles virus (MeV)antigenic polypeptides or immunogenic fragments thereof, andBetacoronavirus (BetaCoV) antigenic polypeptides or immunogenicfragments thereof.2. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof and a PIV3antigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a hMPV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a PIV3 antigenicpolypeptide or an immunogenic fragment thereof.

3. The respiratory virus vaccine of paragraph 2, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the PIV3 antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 12-13 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 12-13.4. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof and a RSVantigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a hMPV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof.

5. The respiratory virus vaccine of paragraph 4, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8.6. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof and MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a hMPV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof.

7. The respiratory virus vaccine of paragraph 6, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the MeV antigenic polypeptide comprises an aminoacid sequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50.8. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof and aBetaCoV antigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a hMPV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a BetaCoV antigenicpolypeptide or an immunogenic fragment thereof.

9. The respiratory virus vaccine of paragraph 8, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 24-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 24-34.10. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof and a RSVantigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a PIV3 antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof.

11. The respiratory virus vaccine of paragraph 10, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13.12. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a PIV3 antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof.

13. The respiratory virus vaccine of paragraph 12, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, and/or wherein the MeV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 47-50 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 47-50.14. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof and aBetaCoV antigenic

polypeptide or an immunogenic fragment thereof; or at least two RNApolynucleotides, one having an open reading frame encoding a PIV3antigenic polypeptide or an immunogenic fragment thereof and one havingan open reading frame encoding a BetaCoV antigenic polypeptide or animmunogenic fragment thereof.

15. The respiratory virus vaccine of paragraph 14, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 24-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 24-34.16. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aRSV antigenic polypeptide or an immunogenic fragment thereof and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a RSV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof.

17. The respiratory virus vaccine of paragraph 16, wherein the MeVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 47-50 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 47-50.18. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aRSV antigenic polypeptide or an immunogenic fragment thereof and aBetaCoV antigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a RSV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a BetaCoV antigenicpolypeptide or an immunogenic fragment thereof.

19. The respiratory virus vaccine of paragraph 18, wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 24-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 24-34.20. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aMeV antigenic polypeptide or an immunogenic fragment thereof and aBetaCoV antigenic polypeptide or an immunogenic fragment thereof; or

at least two RNA polynucleotides, one having an open reading frameencoding a MeV antigenic polypeptide or an immunogenic fragment thereofand one having an open reading frame encoding a BetaCoV antigenicpolypeptide or an immunogenic fragment thereof.

21. The respiratory virus vaccine of paragraph 20, wherein the MeVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 47-50 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 47-50, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 24-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 24-34.22. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, and a RSVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a PIV3 antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a RSV antigenic polypeptide or an immunogenicfragment thereof.

23. The respiratory virus vaccine of paragraph 22, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the PIV3 antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 12-13 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 12-13.24. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a PIV3 antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a MeV antigenic polypeptide or an immunogenicfragment thereof.

25. The respiratory virus vaccine of paragraph 24, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13, and/or wherein the MeVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 47-50 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 47-50.26. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a PIV3 antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

27. The respiratory virus vaccine of paragraph 26, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13 and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 23-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 23-34.28. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a MeV antigenic polypeptide or an immunogenicfragment thereof.

29. The respiratory virus vaccine of paragraph 28, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the MeV antigenic polypeptide comprises an aminoacid sequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50.30. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

31. The respiratory virus vaccine of paragraph 30, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 23-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 23-34.32. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a hMPV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

33. The respiratory virus vaccine of paragraph 32, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the MeV antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50, and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 23-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 23-34.34. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a PIV3 antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a MeV antigenic polypeptide or an immunogenicfragment thereof.

35. The respiratory virus vaccine of paragraph 34, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, and/or wherein the MeV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 47-50 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 47-50.36. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a PIV3 antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a RSV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

37. The respiratory virus vaccine of paragraph 36, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 23-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 23-34.38. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aRSV antigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a RSV antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

39. The respiratory virus vaccine of paragraph 38, wherein the MeVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 47-50 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 47-50, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 23-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 23-34.40. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two or three RNA polynucleotides, one having an open readingframe encoding a PIV3 antigenic polypeptide or an immunogenic fragmentthereof, one having an open reading frame encoding a MeV antigenicpolypeptide or an immunogenic fragment thereof, and one having an openreading frame encoding a BetaCoV antigenic polypeptide or an immunogenicfragment thereof.

41. The respiratory virus vaccine of paragraph 40, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, wherein the MeV antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50, and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 23-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 23-34.42. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a MeVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three or four RNA polynucleotides, one having an openreading frame encoding a hMPV antigenic polypeptide or an immunogenicfragment thereof, one having an open reading frame encoding a PIV3antigenic polypeptide or an immunogenic fragment thereof, one having anopen reading frame encoding a RSV antigenic polypeptide or animmunogenic fragment thereof, and one having an open reading frameencoding a MeV antigenic polypeptide or an immunogenic fragment thereof.

43. The respiratory virus vaccine of paragraph 42, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13, and/or wherein the MeVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 47-50 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 47-50.44. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three or four RNA polynucleotides, one having an openreading frame encoding a hMPV antigenic polypeptide or an immunogenicfragment thereof, one having an open reading frame encoding a PIV3antigenic polypeptide or an immunogenic fragment thereof, one having anopen reading frame encoding a RSV antigenic polypeptide or animmunogenic fragment thereof, and one having an open reading frameencoding a BetaCoV antigenic polypeptide or an immunogenic fragmentthereof.

45. The respiratory virus vaccine of paragraph 44, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13, and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 24-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 24-34.46. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three or four RNA polynucleotides, one having an openreading frame encoding a hMPV antigenic polypeptide or an immunogenicfragment thereof, one having an open reading frame encoding a PIV3antigenic polypeptide or an immunogenic fragment thereof, one having anopen reading frame encoding a MeV antigenic polypeptide or animmunogenic fragment thereof, and one having an open reading frameencoding a BetaCoV antigenic polypeptide or an immunogenic fragmentthereof.

47. The respiratory virus vaccine of paragraph 46, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13, wherein the MeV antigenicpolypeptide comprises an amino acid sequence identified by any one ofSEQ ID NO: 47-50 or an amino acid sequence having at least 90% or 95%identity to an amino acid sequence identified by any one of SEQ ID NO:47-50, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 24-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 24-34.48. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three or four RNA polynucleotides, one having an openreading frame encoding a hMPV antigenic polypeptide or an immunogenicfragment thereof, one having an open reading frame encoding a RSVantigenic polypeptide or an immunogenic fragment thereof, one having anopen reading frame encoding a MeV antigenic polypeptide or animmunogenic fragment thereof, and one having an open reading frameencoding a BetaCoV antigenic polypeptide or an immunogenic fragmentthereof.

49. The respiratory virus vaccine of paragraph 48, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the MeV antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50, and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 24-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 24-34.50. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding aPIV3 antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three or four RNA polynucleotides, one having an openreading frame encoding a PIV3 antigenic polypeptide or an immunogenicfragment thereof, one having an open reading frame encoding a RSVantigenic polypeptide or an immunogenic fragment thereof, one having anopen reading frame encoding a MeV antigenic polypeptide or animmunogenic fragment thereof, and one having an open reading frameencoding a BetaCoV antigenic polypeptide or an immunogenic fragmentthereof.

51. The respiratory virus vaccine of paragraph 50, wherein the PIV3antigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 12-13 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 12-13, wherein the MeV antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 47-50 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 47-50, and/or wherein the BetaCoVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 24-34 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 24-34.52. The respiratory virus vaccine of paragraph 1, comprising:

at least one RNA polynucleotide having an open reading frame encoding ahMPV antigenic polypeptide or an immunogenic fragment thereof, a PIV3antigenic polypeptide or an immunogenic fragment thereof, a RSVantigenic polypeptide or an immunogenic fragment thereof, a MeVantigenic polypeptide or an immunogenic fragment thereof, and a BetaCoVantigenic polypeptide or an immunogenic fragment thereof; or

at least two, three, four or five RNA polynucleotides, one having anopen reading frame encoding a hMPV antigenic polypeptide or animmunogenic fragment thereof, one having an open reading frame encodinga PIV3 antigenic polypeptide or an immunogenic fragment thereof, onehaving an open reading frame encoding a RSV antigenic polypeptide or animmunogenic fragment thereof, one having an open reading frame encodinga MeV antigenic polypeptide or an immunogenic fragment thereof, and onehaving an open reading frame encoding a BetaCoV antigenic polypeptide oran immunogenic fragment thereof.

53. The respiratory virus vaccine of paragraph 52, wherein the hMPVantigenic polypeptide comprises an amino acid sequence identified by anyone of SEQ ID NO: 5-8 or an amino acid sequence having at least 90% or95% identity to an amino acid sequence identified by any one of SEQ IDNO: 5-8, wherein the PIV3 antigenic polypeptide comprises an amino acidsequence identified by any one of SEQ ID NO: 12-13 or an amino acidsequence having at least 90% or 95% identity to an amino acid sequenceidentified by any one of SEQ ID NO: 12-13, wherein the MeV antigenicpolypeptide comprises an amino acid sequence identified by any one ofSEQ ID NO: 47-50 or an amino acid sequence having at least 90% or 95%identity to an amino acid sequence identified by any one of SEQ ID NO:47-50, and/or wherein the BetaCoV antigenic polypeptide comprises anamino acid sequence identified by any one of SEQ ID NO: 24-34 or anamino acid sequence having at least 90% or 95% identity to an amino acidsequence identified by any one of SEQ ID NO: 24-34.54. The vaccine of any one of paragraphs 1-53, wherein at least one RNApolynucleotide has less than 80% identity to wild-type mRNA sequence.55. The vaccine of any one of paragraphs 1-53, wherein at least one RNApolynucleotide has at least 80% identity to wild-type mRNA sequence, butdoes not include wild-type mRNA sequence.56. The vaccine of any one of paragraphs 1-55, wherein at least oneantigenic polypeptide has membrane fusion activity, attaches to cellreceptors, causes fusion of viral and cellular membranes, and/or isresponsible for binding of the virus to a cell being infected.57. The vaccine of any one of paragraphs 1-56, wherein at least one RNApolynucleotide comprises at least one chemical modification.58. The vaccine of paragraph 57, wherein the chemical modification isselected from pseudouridine, N1-methylpseudouridine,N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine and 2′-O-methyl uridine.59. The vaccine of paragraph 57 or 58, wherein the chemical modificationis in the 5-position of the uracil.60. The vaccine of any one of paragraphs 57-59, wherein the chemicalmodification is a N1-methylpseudouridine or N1-ethylpseudouridine.61. The vaccine of any one of paragraphs 57-60, wherein at least 80%, atleast 90% or 100% of the uracil in the open reading frame have achemical modification.62. The vaccine of any one of paragraphs 1-61, wherein at least one RNApolynucleotide further encodes at least one 5′ terminal cap, optionallywherein the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.63. The vaccine of any one of paragraphs 1-62, wherein at least oneantigenic polypeptide or immunogenic fragment thereof is fused to asignal peptide selected from: a HuIgGk signal peptide(METPAQLLFLLLLWLPDTTG; SEQ ID NO: 15); IgE heavy chain epsilon-1 signalpeptide (MDWTWILFLVAAATRVHS; SEQ ID NO: 16); Japanese encephalitis PRMsignal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 17), VSVg proteinsignal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 18) and Japaneseencephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO:19).64. The vaccine of paragraph 63, wherein the signal peptide is fused tothe N-terminus or the C-terminus of at least one antigenic polypeptide.65. The vaccine of any one of paragraphs 1-64, wherein the antigenicpolypeptide or immunogenic fragment thereof comprises a mutated N-linkedglycosylation site.66. The vaccine of any one of paragraphs 1-65 formulated in ananoparticle, optionally a lipid nanoparticle.67. The vaccine of paragraph 66, wherein the lipid nanoparticlecomprises a cationic lipid, a PEG-modified lipid, a sterol and anon-cationic lipid; optionally wherein the lipid nanoparticle carriercomprises a molar ratio of about 20-60% cationic lipid, 0.5-15%PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid;optionally wherein the cationic lipid is an ionizable cationic lipid andthe non-cationic lipid is a neutral lipid, and the sterol is acholesterol; and optionally wherein the cationic lipid is selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Formula (II)68. The vaccine of paragraph 66 or 67, wherein the nanoparticle (e.g.,lipid nanoparticle) comprises a compound of Formula (I) and/or Formula(II), optionally Compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or122.69. The vaccine of any one of paragraphs 1-68 further comprising anadjuvant, optionally a flagellin protein or peptide that optionallycomprises an amino acid sequence identified by any one of SEQ ID NO:54-56.70. The vaccine of any one of paragraphs 1-69, wherein the open readingframe is codon-optimized.71. The vaccine of any one of paragraphs 1-70 formulated in an effectiveamount to produce an antigen-specific immune response.72. A method of inducing an immune response in a subject, the methodcomprising administering to the subject the vaccine of any one ofparagraphs 1-71 in an amount effective to produce an antigen-specificimmune response in the subject.73. The method of paragraph 72, wherein the subject is administered asingle dose of the vaccine, or wherein the subject is administered afirst dose and then a booster dose of the vaccine.74. The method of paragraph 72 or 73, wherein the vaccine isadministered to the subject by intradermal injection or intramuscularinjection.75. The method of any one of paragraphs 72-74, wherein an anti-antigenicpolypeptide antibody titer produced in the subject is increased by atleast 1 log relative to a control, and/or wherein the anti-antigenicpolypeptide antibody titer produced in the subject is increased at least2 times relative to a control.76. The method of any one of paragraphs 72-75, wherein the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasnot been administered a vaccine against the virus, and/or wherein thecontrol is an anti-antigenic polypeptide antibody titer produced in asubject who has been administered a live attenuated vaccine or aninactivated vaccine against the virus, and/or, wherein the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasbeen administered a recombinant protein vaccine or purified proteinvaccine against the virus, and/or wherein the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasbeen administered a VLP vaccine against the virus.77. The method of any one of paragraphs 72-76, wherein the effectiveamount is a dose equivalent to an at least 2-fold reduction in thestandard of care dose of a recombinant protein vaccine or a purifiedprotein vaccine against the virus, and wherein an anti-antigenicpolypeptide antibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant protein vaccineor a purified protein vaccine against the virus, respectively; and/orwherein the effective amount is a dose equivalent to an at least 2-foldreduction in the standard of care dose of a live attenuated vaccine oran inactivated vaccine against the virus, and wherein an anti-antigenicpolypeptide antibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a live attenuated vaccine oran inactivated vaccine against the virus, respectively; and/or whereinthe effective amount is a dose equivalent to an at least 2-foldreduction in the standard of care dose of a VLP vaccine against thevirus, and wherein an anti-antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a VLP vaccine against the virus.78. The method of any one of paragraphs 72-77, wherein the effectiveamount is a total dose of 50 μg-1000 μg, optionally wherein theeffective amount is a dose of 25 μg, 100 μg, 400 μg, or 500 μgadministered to the subject a total of two times.79. The method of any one of paragraphs 72-78, wherein the efficacy ofthe vaccine against the virus is greater than 65%; and/or wherein thevaccine immunizes the subject against the virus for up to 2 years orwherein the vaccine immunizes the subject against the virus for morethan 2 years.80. The method of any one of paragraphs 72-79, wherein the subject hasan age of about 5 years old or younger or wherein the subject has an ageof about 60 years old or older; and/or wherein the subject has a chronicpulmonary disease; and/or the subject has been exposed to the virus,wherein the subject is infected with the virus, or wherein the subjectis at risk of infection by the virus; and/or wherein the subject isimmunocompromised.81. The respiratory virus vaccine of any one of paragraphs 1-71,comprising at least one (e.g., at least two, at least three, at leastfour, or at least five) RNA polynucleotide having an open reading frameencoding at least one (e.g., at least two, at least three, at leastfour, or at least five) antigenic polypeptide selected from hMPVantigenic polypeptides (SEQ ID NO: 5-8), PIV3 antigenic polypeptides(SEQ ID NO: 12-13), RSV antigenic polypeptides, MeV antigenicpolypeptides (SEQ ID NO: 47-50) and BetaCoV antigenic polypeptides(e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH or HCoV-HKU1; (SEQ ID NO: 24-34)), formulated in a cationiclipid nanoparticle

(a) having a molar ratio of about 20-60% cationic lipid, about 5-25%non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modifiedlipid, and/or

(b) comprising a compound of Formula (I) and/or Formula (II),

wherein the at least one (e.g., at least two, at least three, at leastfour, or at least five) RNA polynucleotide comprises at least onechemical modification.

82. The respiratory virus vaccine of any one of paragraphs 1-71,comprising at least one (e.g., at least two, at least three, at leastfour, or at least five) RNA polynucleotide having an open reading frameencoding at least one (e.g., at least two, at least three, at leastfour, or at least five) antigenic polypeptide selected from hMPVantigenic polypeptides (SEQ ID NO: 5-8), PIV3 antigenic polypeptides(SEQ ID NO: 12-13), RSV antigenic polypeptides, MeV antigenicpolypeptides (SEQ ID NO: 47-50) and BetaCoV antigenic polypeptides(e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH or HCoV-HKU1; (SEQ ID NO: 24-34)), formulated in a cationiclipid nanoparticle

(a) having a molar ratio of about 20-60% cationic lipid, about 5-25%non-cationic lipid, about 25-55% sterol, and about 0.5-15% PEG-modifiedlipid, and/or

(b) comprising at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14) Compound selected from Compounds 3, 18, 20, 25,26, 29, 30, 60, 108-112 and 122.

83. The respiratory virus vaccine of paragraphs 81 or 82, wherein the atleast one antigenic polypeptide is selected from hMPV antigenticpolypeptides (e.g., SEQ ID NO: 5-8).

84. The respiratory virus vaccine of any one of paragraphs 81-83,wherein the at least one antigenic polypeptide is selected from PIV3antigentic polypeptides (e.g., SEQ ID NO: 12-13).

85. The respiratory virus vaccine of any one of paragraphs 81-84,wherein the at least one antigenic polypeptide is selected from RSVantigentic polypeptides.

86. The respiratory virus vaccine of any one of paragraphs 81-85,wherein the at least one antigenic polypeptide is selected from MeVantigentic polypeptides (e.g., SEQ ID NO: 47-50).

87. The respiratory virus vaccine of any one of paragraphs 81-86,wherein the at least one antigenic polypeptide is selected from BetaCoVantigentic polypeptides (e.g., SEQ ID NO: 24-34).

88. The respiratory virus vaccine of paragraph 87, wherein the BetaCoVantigentic polypeptides are MERS antigentic polypeptides.

89. The respiratory virus vaccine of paragraph 87, wherein the BetaCoVantigentic polypeptides are SARS antigentic polypeptides.

90. The respiratory virus vaccine of any one of paragraphs 81-89,wherein the at least one (e.g., at least two, at least three, at leastfour, or at least five) RNA polynucleotide comprises at least onechemical modification (e.g., selected from pseudouridine,N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine).91. A respiratory virus vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap, an open reading frame encoding at least one respiratoryvirus antigenic polypeptide, and a 3′ polyA tail.

92. The vaccine of paragraph 91, wherein the at least one mRNApolynucleotide comprises a sequence identified by any one of SEQ ID NO:57-80.

93. The vaccine of paragraph 91 or 92, wherein the 5′ terminal cap is orcomprises 7mG(5′)ppp(5′)NlmpNp.

94. The vaccine of any one of paragraphs 91-93, wherein 100% of theuracil in the open reading frame is modified to include N1-methylpseudouridine at the 5-position of the uracil.

95. The vaccine of any one of paragraphs 91-94, wherein the vaccine isformulated in a lipid nanoparticle comprising: DLin-MC3-DMA;cholesterol; 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); andpolyethylene glycol (PEG)2000-DMG.

96. The vaccine of paragraph 95, wherein the lipid nanoparticle furthercomprises trisodium citrate buffer, sucrose and water.

97. A respiratory syncytial virus (RSV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by any oneof SEQ ID NO: 57-80 and a 3′ polyA tail, formulated in a lipidnanoparticle comprising DLin-MC3-DMA, cholesterol,1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and polyethyleneglycol (PEG)2000-DMG, wherein the uracil nucleotides of the sequenceidentified by any one of SEQ ID NO: 57-80 are modified to includeN1-methyl pseudouridine at the 5-position of the uracil nucleotide.

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The disclosure iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1: Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand/or parts or regions thereof may be accomplished utilizing themethods taught in International Publication WO2014/152027, entitled“Manufacturing Methods for Production of RNA Transcripts,” the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalPublication WO2014/152030 and International Publication WO2014/152031,each of which is incorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in International Publication WO2014/144039, which isincorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using polynucleotide mapping, reverse transcriptasesequencing, charge distribution analysis, detection of RNA impurities,or any combination of two or more of the foregoing. “Characterizing”comprises determining the RNA transcript sequence, determining thepurity of the RNA transcript, or determining the charge heterogeneity ofthe RNA transcript, for example. Such methods are taught in, forexample, International Publication WO2014/144711 and InternationalPublication WO2014/144767, the content of each of which is incorporatedherein by reference in its entirety.

Example 2: Chimeric Polynucleotide Synthesis

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry. Afirst region or part of 100 nucleotides or less is chemicallysynthesized with a 5′ monophosphate and terminal 3′desOH or blocked OH,for example. If the region is longer than 80 nucleotides, it may besynthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

For ligation methods, ligation with DNA T4 ligase, followed by treatmentwith DNase should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part may comprise a phosphate-sugarbackbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide may be made using a series of startingsegments. Such segments include:

(a) a capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) a 5′ triphosphate segment, which may include the coding region of apolypeptide and a normal 3′OH (SEG. 2)

(c) a 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) may be treatedwith cordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG. 2-SEG. 3 construct may thenbe purified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA may be performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 μM) 0.75 μl;Reverse Primer (10 μM) 0.75 μl; Template cDNA 100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions may be at 95° C. for 5 min. Thereaction may be performed for 25 cycles of 98° C. for 20 sec, then 58°C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min, then 4° C.to termination.

The reaction may be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions may require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA may be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thatthe cDNA is the expected size. The cDNA may then be submitted forsequencing analysis before proceeding to the in vitro transcriptionreaction.

Example 4: In Vitro Transcription (IVT)

The in vitro transcription reaction generates RNA polynucleotides. Suchpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure, including chemically modified RNA (e.g., mRNA)polynucleotides. The chemically modified RNA polynucleotides can beuniformly modified polynucleotides. The in vitro transcription reactionutilizes a custom mix of nucleotide triphosphates (NTPs). The NTPs maycomprise chemically modified NTPs, or a mix of natural and chemicallymodified NTPs, or natural NTPs.

A typical in vitro transcription reaction includes the following:

1) Template cDNA 1.0 μg 2) 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3) CustomNTPs (25 mM each) 0.2 μl 4) RNase Inhibitor 20 U 5) T7 RNA polymerase3000 U 6) dH₂0 up to 20.0 μl. and 7) Incubation at 37° C. for 3 hr-5hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase may then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA may bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA polynucleotide may be quantified usingthe NanoDrop and analyzed by agarose gel electrophoresis to confirm theRNA polynucleotide is the proper size and that no degradation of the RNAhas occurred.

Example 5: Enzymatic Capping

Capping of a RNA polynucleotide is performed as follows where themixture includes: IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixtureis incubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The RNA polynucleotide may then be purified using Ambion's MEGACLEAR™Kit (Austin, Tex.) following the manufacturer's instructions. Followingthe cleanup, the RNA may be quantified using the NANODROP™(ThermoFisher, Waltham, Mass.) and analyzed by agarose gelelectrophoresis to confirm the RNA polynucleotide is the proper size andthat no degradation of the RNA has occurred. The RNA polynucleotideproduct may also be sequenced by running a reverse-transcription-PCR togenerate the cDNA for sequencing.

Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingcapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymerasemay be a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence, polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe present disclosure.

Example 7. Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 8: Capping Assays

Protein Expression Assay

Polynucleotides (e.g., mRNA) encoding a polypeptide, containing any ofthe caps taught herein, can be transfected into cells at equalconcentrations. The amount of protein secreted into the culture mediumcan be assayed by ELISA at 6, 12, 24 and/or 36 hours post-transfection.Synthetic polynucleotides that secrete higher levels of protein into themedium correspond to a synthetic polynucleotide with a highertranslationally-competent cap structure.

Purity Analysis Synthesis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. RNA polynucleotideswith a single, consolidated band by electrophoresis correspond to thehigher purity product compared to polynucleotides with multiple bands orstreaking bands. Chemically modified RNA polynucleotides with a singleHPLC peak also correspond to a higher purity product. The cappingreaction with a higher efficiency provides a more pure polynucleotidepopulation.

Cytokine Analysis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be transfected into cells at multipleconcentrations. The amount of pro-inflammatory cytokines, such asTNF-alpha and IFN-beta, secreted into the culture medium can be assayedby ELISA at 6, 12, 24 and/or 36 hours post-transfection. RNApolynucleotides resulting in the secretion of higher levels ofpro-inflammatory cytokines into the medium correspond to apolynucleotides containing an immune-activating cap structure.

Capping Reaction Efficiency

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be analyzed for capping reactionefficiency by LC-MS after nuclease treatment. Nuclease treatment ofcapped polynucleotides yield a mixture of free nucleotides and thecapped 5′-5-triphosphate cap structure detectable by LC-MS. The amountof capped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and correspond to cappingreaction efficiency. The cap structure with a higher capping reactionefficiency has a higher amount of capped product by LC-MS.

Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual RNA polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) may be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes, according to the manufacturer protocol.

Example 10: Nanodrop Modified RNA Quantification and UV Spectral Data

Chemically modified RNA polynucleotides in TE buffer (1 μl) are used forNanodrop UV absorbance readings to quantitate the yield of eachpolynucleotide from an chemical synthesis or in vitro transcriptionreaction.

Example 11: Formulation of Modified mRNA Using Lipidoids

RNA (e.g., mRNA) polynucleotides may be formulated for in vitroexperiments by mixing the polynucleotides with the lipidoid at a setratio prior to addition to cells. In vivo formulation may require theaddition of extra ingredients to facilitate circulation throughout thebody. To test the ability of these lipidoids to form particles suitablefor in vivo work, a standard formulation process used for siRNA-lipidoidformulations may be used as a starting point. After formation of theparticle, polynucleotide is added and allowed to integrate with thecomplex. The encapsulation efficiency is determined using a standard dyeexclusion assays.

Example 12: Immunogenicity Study

The instant study is designed to test the immunogenicity in mice ofcandidate hMPV vaccines comprising a mRNA polynucleotide encoding Fusion(F) glycoprotein, major surface glycoprotein G, or a combinationthereof, obtained from hMPV.

Mice are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) with candidate vaccines. Candidate vaccines arechemically modified or unmodified. A total of four immunizations aregiven at 3-week intervals (i.e., at weeks 0, 3, 6, and 9), and sera arecollected after each immunization until weeks 33-51. Serum antibodytiters against Fusion (F) glycoprotein or major surface glycoprotein (G)protein are determined by ELISA. Sera collected from each mouse duringweeks 10-16 are pooled, and total IgG purified. Purified antibodies areused for immunoelectron microscopy, antibody-affinity testing, and invitro protection assays.

Example 13: hMPV Rodent Challenge

The instant study is designed to test the efficacy in cotton rats ofcandidate hMPV vaccines against a lethal challenge using an hMPV vaccinecomprising mRNA encoding Fusion (F) glycoprotein, major surfaceglycoprotein G, or a combination of both antigens obtained from hMPV.Cotton rats are challenged with a lethal dose of the hMPV.

Animals are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) at week 0 and week 3 with candidate hMPV vaccineswith and without adjuvant. Candidate vaccines are chemically modified orunmodified. The animals are then challenged with a lethal dose of hMPVon week 7 via IV, IM or ID. Endpoint is day 13 post infection, death oreuthanasia. Animals displaying severe illness as determined by >30%weight loss, extreme lethargy or paralysis are euthanized. Bodytemperature and weight are assessed and recorded daily.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid isDLin-KC2-DMA (50 mol %) or DLin-MC3-DMA (50 mol %), the non-cationiclipid is DSPC (10 mol %), the PEG lipid is PEG-DOMG (1.5 mol %) and thestructural lipid is cholesterol (38.5 mol %), for example.

Example 14: Immunogenicity of hMPV mRNA Vaccine in BALB/c Mice

The instant study was designed to test the immunogenicity in BALB/c miceof hMPV vaccines comprising an mRNA polynucleotide encoding the hMPVFusion (F) glycoprotein. The mRNA polynucleotide encodes the full-lengthfusion protein and comprises the wild-type nucleotide sequence obtainedfrom the hMPV A2a strain. Mice were divided into 3 groups (n=8 for eachgroup) and immunized intramuscularly (IM) with PBS, a 10 μg dose of mRNAvaccines encoding hMPV fusion protein, or a 2 μg dose of mRNA vaccinesencoding hMPV fusion protein. A total of two immunizations were given at3-week intervals (i.e., at weeks 0, and 3 weeks), and sera werecollected after each immunization according to the schedule described inTable 1. Serum antibody titers against hMPV fusion glycoprotein weredetermined by ELISA and antibodies were detected in the sera collectedon day 14 onward. Both vaccine doses tested induced comparable levels ofimmune response in mice (FIGS. 2A-2C).

Additionally, mice sera were used for IgG isotyping (FIGS. 3A-3C). BothhMPV fusion protein-specific IgG1 and IgG2a were detected in mice sera.hMPV fusion protein mRNA vaccine also induced Th1 and Th2 cytokineresponses, with a Th1 bias.

Sera from mice immunized with either 10 μg or 2 μg doses of the hMPVfusion protein mRNA vaccine contain neutralizing antibodies. The abilityof these antibodies to neutralize hMPV B2 strain was also tested. Theantibody-containing sera successfully neutralized the hMPV B2 virus(FIG. 4).

Example 15: T-Cell Stimulation

The instant study was designed to test T-cell stimulation in thesplenocytes of mice immunized with mRNA vaccines encoding hMPV fusionprotein, as described herein. Immunization of BALB/c mice was performedas described in Example 14. The splenocytes for each group were pooledand split into two parts. One part of splenocytes from each group ofmice was stimulated with hMPV-free media, Concanavalin A or a hMPVfusion protein peptide pool comprising 15-mers (15 amino acids long);while the other part of splenocytes from each group of mice wasstimulated with hMPV-free media, Concanavalin A or inactivated hMPVvirus. Secreted mouse cytokines were measured using the Meso ScaleDiscovery (MSD) assay.

Cytokines specific to Th1 or Th2 responses were measured. For Th1response, IFN-γ, IL2 and IL12 were detected from splenocytes stimulatedwith the hMPV fusion protein peptide pool at a level comparable to thatof Concanavalin A (FIGS. 5A-5C). For a Th2 response, the hMPV fusionprotein peptide pool induced the secretion of detectable IL10, TNF-α,IL4 and IL, but not IL5, while Concanavalin A stimulated the secretionof all the above-mentioned Th2 cytokines (FIGS. 6A-6E) at a much higherlevel.

In contrast, inactivated hMPV virus only induced the secretion of IL2 inthe Th1 response comparable to that of Concanavalin A (FIGS. 7A-7C). Forthe Th2 response, the inactivated hMPV virus induced the secretion ofdetectable IL10, TNF-α, IL4 and IL6, but not IL5, while Concanavalin Astimulated the secretion of all the above-mentioned Th2 cytokines (FIGS.8A-8E) at a much higher level.

Example 16: hMPV Rodent Challenge in Cotton Rats Immunized with mRNAVaccine Encoding hMPV Fusion Protein

The instant study was designed to test the efficacy in cotton rats ofhMPV vaccines against a lethal challenge. mRNA vaccines encoding hMPVfusion protein were used. The mRNA polynucleotide encodes a full-lengthfusion protein and comprises the wild-type nucleotide sequence obtainedfrom the hMPV A2a strain.

Cotton rats were immunized intramuscularly (IM) at week 0 and week 3with the mRNA vaccines encoding hMPV fusion protein with either 2 μg or10 μg doses for each immunization. The animals were then challenged witha lethal dose of hMPV in week 7 post initial immunization via IV, IM orID. The endpoint was day 13 post infection, death or euthanasia. Viraltiters in the noses and lungs of the cotton rats were measured. Theresults (FIGS. 9A and 9B) show that a 10 μg dose of mRNA vaccineprotected the cotton mice 100% in the lung and drastically reduced theviral titer in the nose after challenge (˜2 log reduction). Moreover, a2 μg dose of mRNA vaccine showed a 1 log reduction in lung viral titerin the cotton mice challenged.

Further, the histopathology of the lungs of the cotton mice immunizedand challenged showed no pathology associated with vaccine-enhanceddisease (FIG. 10).

Example 17. Immunogenicity Study

The instant study is designed to test the immunogenicity in mice ofcandidate PIV3 vaccines comprising a mRNA polynucleotide encodinghemagglutinin-neuraminidase or fusion protein (F or F0) obtained fromPIV3.

Mice are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) with candidate vaccines. Candidate vaccines arechemically modified or unmodified. A total of four immunizations aregiven at 3-week intervals (i.e., at weeks 0, 3, 6, and 9), and sera arecollected after each immunization until weeks 33-51. Serum antibodytiters against hemagglutinin-neuraminidase or fusion protein (F or F0)are determined by ELISA. Sera collected from each mouse during weeks10-16 are, optionally, pooled, and total IgGs are purified. Purifiedantibodies are used for immunoelectron microscopy, antibody-affinitytesting, and in vitro protection assays.

Example 18: PIV3 Rodent Challenge

The instant study is designed to test the efficacy in cotton rats ofcandidate PIV3 vaccines against a lethal challenge using a PIV3 vaccinecomprising mRNA encoding hemagglutinin-neuraminidase or fusion protein(F or F0) obtained from PIV3. Cotton rats are challenged with a lethaldose of the PIV3.

Animals are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) at week 0 and week 3 with candidate PIV3 vaccineswith and without adjuvant. Candidate vaccines are chemically modified orunmodified. The animals are then challenged with a lethal dose of PIV3on week 7 via IV, IM or ID. Endpoint is day 13 post infection, death oreuthanasia. Animals displaying severe illness as determined by >30%weight loss, extreme lethargy or paralysis are euthanized. Bodytemperature and weight are assessed and recorded daily.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid isDLin-KC2-DMA (50 mol %) or DLin-MC3-DMA (50 mol %), the non-cationiclipid is DSPC (10 mol %), the PEG lipid is PEG-DOMG (1.5 mol %) and thestructural lipid is cholesterol (38.5 mol %), for example.

Example 19: hMPV/PIV Cotton Rat Challenge

The instant study was designed to test the efficacy in cotton rats ofcandidate hMPV mRNA vaccines, PIV3 mRNA vaccines, or hMPV/PIVcombination mRNA vaccines against a lethal challenge using PIV3 strainor hMPV/A2 strain. The study design is shown in Table 9.

Cotton rats of 10-12 weeks old were divided into 12 groups (n=5), andeach group was vaccinated with mRNA vaccines indicated in Table 9. ThePIV3 vaccine comprises mRNA encoding hemagglutinin-neuraminidase orfusion protein (F or F0) obtained from PIV3. The hMPV mRNA vaccineencodes the full-length hMPV fusion protein. The hMPV/PIV combinationmRNA vaccine is a mixture of the PIV3 vaccine and hMPV vaccine at a 1:1ratio.

Cotton rats were immunized intramuscularly (IM) at week 0 and week 3with candidate vaccines with the doses indicated in Table 9. Cotton ratsimmunized with hMPV mRNA vaccines or hMPV/PIV combination mRNA vaccineswere challenged with a lethal dose of hMPV/A2 strain on week 7 via IM.Cotton rats immunized with PIV mRNA vaccines or hMPV/PIV combinationmRNA vaccines were challenged with a lethal dose of PIV3 strain on week7 via IM.

The endpoint was day 13 post infection, death or euthanasia. Animalsdisplaying severe illness as determined by >30% weight loss, extremelethargy or paralysis were euthanized. Body temperature and weight wereassessed and recorded daily.

Lung and nose hMPV/A2 (FIG. 12) or PIV3 (FIG. 13) viral titers wereassessed. Lung histopathology of the immunized and challenged cotton ratimmunized and challenged were assessed to determine pathology associatedwith vaccine enhance disease. Neutralization antibody titers in theserum of immunized cotton rats on day 0 and 42 post immunization wereassessed (FIG. 11).

hMPV/A2 (FIG. 14) or PIV3 (FIG. 15) neutralizing antibody titers in theserum samples of the immunized cotton rat 42 days post immunization weremeasured. All mRNA vaccines tested induced strong neutralizingantibodies cotton rats. Lung histopathology of the immunized cotton ratswere also evaluated (FIG. 16). Low occurrence of alevolitis andinterstitial pneumonia was observed, indicating no antibody-dependentenhancement (ADE) of hMPV or PIV associated diseases.

Example 20: Betacoronavirus Immunogenicity Study

The instant study is designed to test the immunogenicity in rabbits ofcandidate Betacoronavirus (e.g., MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH or HCoV-HKU1 or a combinationthereof) vaccines comprising a mRNA polynucleotide encoding the spike(S) protein, the S1 subunit (S1) of the spike protein, or the S2 subunit(S2) of the spike protein obtained from a Betacoronavirus (e.g.,MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH orHCoV-HKU1).

Rabbits are vaccinated on week 0 and 3 via intravenous (IV),intramuscular (IM), or intradermal (ID) routes. One group remainsunvaccinated and one is administered inactivated Betacoronavirus. Serumis collected from each rabbit on weeks 1, 3 (pre-dose) and 5. Individualbleeds are tested for anti-S, anti-S1 or anti-S2 activity via a virusneutralization assay from all three time points, and pooled samples fromweek 5 only are tested by Western blot using inactivated Betacoronavirus(e.g., inactivated MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63,HCoV-NL, HCoV-NH or HCoV-HKU1).

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid isDLin-KC2-DMA (50 mol %) or DLin-MC3-DMA (50 mol %), the non-cationiclipid is DSPC (10 mol %), the PEG lipid is PEG-DOMG (1.5 mol %) and thestructural lipid is cholesterol (38.5 mol %), for example.

Example 21: Betacoronavirus Challenge

The instant study is designed to test the efficacy in rabbits ofcandidate Betacoronavirus (e.g., MERS-CoV, SARS-CoV, HCoV-OC43,HCoV-HKU1 or a combination thereof) vaccines against a lethal challengeusing a Betacoronavirus (e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-HKU1or a combination thereof) vaccine comprising mRNA encoding the spike (S)protein, the S1 subunit (S1) of the spike protein, or the S2 subunit(S2) of the spike protein obtained from Betacoronavirus (e.g., MERS-CoV,SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH orHCoV-HKU1). Rabbits are challenged with a lethal dose (10×LD90; ˜100plaque-forming units; PFU) of Betacoronavirus (e.g., MERS-CoV, SARS-CoV,HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH or HCoV-HKU1).

The animals used are 6-8 week old female rabbits in groups of 10.Rabbits are vaccinated on weeks 0 and 3 via an IM, ID or IV route ofadministration. Candidate vaccines are chemically modified orunmodified. Rabbit serum is tested for microneutralization (see Example14). Rabbits are then challenged with ˜1 LD90 of Betacoronavirus (e.g.,MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH orHCoV-HKU1) on week 7 via an IN, IM, ID or IV route of administration.Endpoint is day 13 post infection, death or euthanasia. Animalsdisplaying severe illness as determined by >30% weight loss, extremelethargy or paralysis are euthanized. Body temperature and weight areassessed and recorded daily.

Example 22: Microneutralization Assay

Nine serial 2-fold dilutions (1:50-1:12,800) of rabbit serum are made in50 μl virus growth medium (VGM) with trypsin in 96 well microtiterplates. Fifty microliters of virus containing ˜50 pfu of Betacoronavirus(e.g., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL,HCoV-NH or HCoV-HKU1) is added to the serum dilutions and allowed toincubate for 60 minutes at room temperature (RT). Positive control wellsof virus without sera and negative control wells without virus or seraare included in triplicate on each plate. While the serum-virus mixturesincubate, a single cell suspension of Madin-Darby Canine-Kidney cellsare prepared by trypsinizing (Gibco 0.5% bovine pancrease trypsin inEDTA) a confluent monolayer and suspended cells are transferred to a 50ml centrifuge tube, topped with sterile PBS and gently mixed. The cellsare then pelleted at 200 g for 5 minutes, supernatant aspirated andcells resuspended in PBS. This procedure is repeated once and the cellsare resuspended at a concentration of 3×10⁵/ml in VGM with porcinetrypsin. Then, 100 μl of cells are added to the serum-virus mixtures andthe plates incubated at 35° C. in CO₂ for 5 days. The plates are fixedwith 80% acetone in phosphate buffered saline (PBS) for 15 minutes atRT, air dried and then blocked for 30 minutes containing PBS with 0.5%gelatin and 2% FCS. An antibody to the S proteins, 51 protein or S2protein is diluted in PBS with 0.5% gelatin/2% FCS/0.5% Tween 20 andincubated at RT for 2 hours. Wells are washed and horseradishperoxidase-conjugated goat anti-mouse IgG added, followed by another 2hour incubation. After washing, 0-phenylenediamine dihydrochloride isadded and the neutralization titer is defined as the titer of serum thatreduced color development by 50% compared to the positive control wells.

Example 23: MERS CoV Vaccine Immunogenicity Study in Mice

The instant study was designed to test the immunogenicity in mice ofcandidate MERS-CoV vaccines comprising a mRNA polynucleotide encodingthe full-length Spike (S) protein, or the S2 subunit (S2) of the Spikeprotein obtained from MERS-CoV.

Mice were vaccinated with a 10 μg dose of MERS-CoV mRNA vaccine encodingeither the full-length MERS-CoV Spike (S) protein, or the S2 subunit(S2) of the Spike protein on days 0 and 21. Sera were collected fromeach mice on days 0, 21, 42, and 56. Individual bleeds were tested foranti-S, anti-S2 activity via a virus neutralization assay from all fourtime points.

As shown in FIG. 17, the MERS-CoV vaccine encoding the full-length Sprotein induced strong immune response after the boost dose on day 21.Further, full-length S protein vaccine generated much higherneutralizing antibody titers as compared to S2 alone (FIG. 18).

Example 24: MERS CoV Vaccine Immunogenicity Study in New Zealand WhiteRabbits

The instant study was designed to test the immunogenicity of candidateMERS-CoV mRNA vaccines encoding the full-length Spike (S) protein. TheNew Zealand white rabbits used in this study weighed about 4-5 kg. Therabbits were divided into three groups (Group 1a, Group 1b, and Group 2,n=8). Rabbits in Group 1a were immunized intramuscularly (IM) with one20 μg dose of the MERS-CoV mRNA vaccine encoding the full-length Spikeprotein on day 0. Rabbits in Group 1b were immunized intramuscularly(IM) with one 20 μg dose of the MERS-CoV mRNA vaccine encoding thefull-length Spike protein on day 0, and again on day 21 (booster dose).Group 2 received placebo (PBS). The immunized rabbits were thenchallenged and samples were collected 4 days after challenge. The viralloads in the lungs, bronchoalveolar lavage (Bal), nose, and throat ofthe rabbits were determined, e.g., via quantitative PCR. Replicatingvirus in the lung tissues of the rabbits were also detected. Lunghistopathology were evaluated and the neutralizing antibody titers inserum samples of the rabbits were determined.

Two 20 μg doses of MERS-CoV mRNA vaccine resulted in a 3 log reductionof viral load in the nose and led to complete protection in the throatof the New Zealand white rabbits (FIG. 19A). Two 20 μg doses of MERS-CoVmRNA vaccine also resulted in a 4 log reduction of viral load in the BALof the New Zealand white rabbits (FIG. 19B). One 20 μg dose of MERS-CoVmRNA vaccine resulted in a 2 log reduction of viral load, while two 20μg doses of MERS-CoV mRNA vaccine resulted in an over 4 log reduction ofviral load in the lungs of the New Zealand white rabbits (FIG. 19C).

Quantitative PCR results show that two 20 μg doses of MERS-CoV mRNAvaccine reduced over 99% (2 log) of viruses in the lungs of New Zealandwhite rabbits (FIG. 20A). No replicating virus were detected in thelungs (FIG. 20B).

Further, as shown in FIG. 21, two 20 μg doses of MERS-CoV mRNA vaccineinduced significant amount of neutralizing antibodies against MERS-CoV(EC₅₀ between 500-1000). The MERS-CoV mRNA vaccine induced antibodytiter is 3-5 fold better than any other vaccines tested in the samemodel.

Example 25: Immunogenicity Study

The instant study is designed to test the immunogenicity in mice ofcandidate MeV vaccines comprising a mRNA polynucleotide encoding MeVhemagglutinin (HA) protein, MeV Fusion (F) protein or a combination ofboth.

Mice are immunized intravenously (IV), intramuscularly (IM), orintradermally (ID) with candidate vaccines. Up to three immunizationsare given at 3-week intervals (i.e., at weeks 0, 3, 6, and 9), and seraare collected after each immunization until weeks 33-51. Serum antibodytiters against MeV HA protein or MeV F protein are determined by ELISA.

Example 26: MeV Rodent Challenge

The instant study is designed to test the efficacy in transgenic mice ofcandidate MeV vaccines against a lethal challenge using a MeV vaccinecomprising mRNA encoding MeV HA protein or MeV F protein. The transgenicmice express human receptor CD46 or signaling lymphocyte activationmolecule (SLAM) (also referred to as CD150). Humans are the only naturalhost for MeV infection, thus transgenic lines are required for thisstudy. CD46 is a complement regulatory protein that protects host tissuefrom complement deposition by binding to complement components C3b andC4b. Its expression on murine fibroblast and lymphoid cell lines rendersthese otherwise refractory cells permissive for MeV infection, and theexpression of CD46 on primate cells parallels the clinical tropism ofMeV infection in humans and nonhuman primates (Rall G F et al. PNAS USA1997; 94(9):4659-63). SLAM is a type 1 membrane glycoprotein belongingto the immunoglobulin superfamily. It is expressed on the surface ofactivated lymphocytes, macrophages, and dendritic cells and is thoughtto play an important role in lymphocyte signaling. SLAM is a receptorfor both wild-type and vaccine MeV strains (Sellin C I et al. J Virol.2006; 80(13):6420-29).

CD46 or SLAM/CD150 transgenic mice are challenged with a lethal dose ofthe MeV. Animals are immunized intravenously (IV), intramuscularly (IM),or intradermally (ID) at week 0 and week 3 with candidate MeV vaccineswith and without adjuvant. The animals are then challenged with a lethaldose of MeV on week 7 via IV, IM or ID. Endpoint is day 13 postinfection, death or euthanasia. Animals displaying severe illness asdetermined by >30% weight loss, extreme lethargy or paralysis areeuthanized. Body temperature and weight are assessed and recorded daily.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid isDLin-KC2-DMA (50 mol %), the non-cationic lipid is DSPC (10 mol %), thePEG lipid is PEG-DOMG (1.5 mol %) and the structural lipid ischolesterol (38.5 mol %), for example.

TABLE 1 hMPV Immunogenicity studies bleeding schedule Animal groups Day(n = 8) vaccine −2 0 7 14 21 28 35 56 Placebo Group 1 PBS Pre- PrimeBleeds Bleeds Bleeds/ Bleeds Bleeds Harvest Spleens/ (n = 8) (IM) BleedBoost Terminal Bleeds 10 μg Group 2 10 μg Dose (n = 8) (IM)  2 μg Group3  2 μg Dose (n = 8) (IM) Total n = 24

Each of the sequences described herein encompasses a chemically modifiedsequence or an unmodified sequence which includes no nucleotidemodifications.

TABLE 2  SEQ ID Description Sequence NO: hMPV Nucleic Acid Sequencesgi|122891979|gb| ATGAGCTGGAAGGTGGTGATTATCTTCAGCCTGCTGATTA 1EF051124.1| Human CACCTCAACACGGCCTGAAGGAGAGCTACCTGGAAGAGAmetapneumo virus GCTGCTCCACCATCACCGAGGGCTACCTGAGCGTGCTGC isolate TN/92-4GGACCGGCTGGTACACCAACGTGTTCACCCTGGAGGTGG fusion protein gene,GCGACGTGGAGAACCTGACCTGCAGCGACGGCCCTAGCC complete genomeTGATCAAGACCGAGCTGGACCTGACCAAGAGCGCTCTGAGAGAGCTGAAGACCGTGTCCGCCGACCAGCTGGCCAGAGAGGAACAGATCGAGAACCCTCGGCAGAGCAGATTCGTGCTGGGCGCCATCGCTCTGGGAGTCGCCGCTGCCGCTGCAGTGACAGCTGGAGTGGCCATTGCTAAGACCATCAGACTGGAAAGCGAGGTGACAGCCATCAACAATGCCCTGAAGAAGACCAACGAGGCCGTGAGCACCCTGGGCAATGGAGTGAGAGTGCTGGCCACAGCCGTGCGGGAGCTGAAGGACTTCGTGAGCAAGAACCTGACCAGAGCCATCAACAAGAACAAGTGCGACATCGATGACCTGAAGATGGCCGTGAGCTTCTCCCAGTTCAACAGACGGTTCCTGAACGTGGTGAGACAGTTCTCCGACAACGCTGGAATCACACCTGCCATTAGCCTGGACCTGATGACCGACGCCGAGCTGGCTAGAGCCGTGCCCAACATGCCCACCAGCGCTGGCCAGATCAAGCTGATGCTGGAGAACAGAGCCATGGTGCGGAGAAAGGGCTTCGGCATCCTGATTGGGGTGTATGGAAGCTCCGTGATCTACATGGTGCAGCTGCCCATCTTCGGCGTGATCGACACACCCTGCTGGATCGTGAAGGCCGCTCCTAGCTGCTCCGAGAAGAAAGGAAACTATGCCTGTCTGCTGAGAGAGGACCAGGGCTGGTACTGCCAGAACGCCGGAAGCACAGTGTACTATCCCAACGAGAAGGACTGCGAGACCAGAGGCGACCACGTGTTCTGCGACACCGCTGCCGGAATCAACGTGGCCGAGCAGAGCAAGGAGTGCAACATCAACATCAGCACAACCAACTACCCCTGCAAGGTGAGCACCGGACGGCACCCCATCAGCATGGTGGCTCTGAGCCCTCTGGGCGCTCTGGTGGCCTGCTATAAGGGCGTGTCCTGTAGCATCGGCAGCAATCGGGTGGGCATCATCAAGCAGCTGAACAAGGGATGCTCCTACATCACCAACCAGGACGCCGACACCGTGACCATCGACAACACCGTGTACCAGCTGAGCAAGGTGGAGGGCGAGCAGCACGTGATCAAGGGCAGACCCGTGAGCTCCAGCTTCGACCCCATCAAGTTCCCTGAGGACCAGTTCAACGTGGCCCTGGACCAGGTGTTTGAGAACATCGAGAACAGCCAGGCCCTGGTGGACCAGAGCAACAGAATCCTGTCCAGCGCTGAGAAGGGCAACACCGGCTTCATCATTGTGATCATTCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGAGCATCTTCATCATTATCAAGAAGACCAAGAAACCCACCGGAGCCCCTCCTGAGCTGAGCGGCGTGACCAACAAT GGCTTCATTCCCCACAACTGAgb|AY525843.1|:  ATGTCTTGGAAAGTGATGATCATCATTTCGTTACTCATAA 23065-4684 Human CACCCCAGCACGGGCTAAAGGAGAGTTATTTGGAAGAAT metapneumo virusCATGTAGTACTATAACTGAGGGATACCTCAGTGTTTTAAG isolate NL/1/99,AACAGGCTGGTACACTAATGTCTTCACATTAGAAGTTGGT complete genomeGATGTTGAAAATCTTACATGTACTGATGGACCTAGCTTAATCAAAACAGAACTTGATCTAACAAAAAGTGCTTTAAGGGAACTCAAAACAGTCTCTGCTGATCAGTTGGCGAGAGAGGAGCAAATTGAAAATCCCAGACAATCAAGATTTGTCTTAGGTGCGATAGCTCTCGGAGTTGCTACAGCAGCAGCAGTCACAGCAGGCATTGCAATAGCCAAAACCATAAGGCTTGAGAGTGAGGTGAATGCAATTAAAGGTGCTCTCAAACAAACTAATGAAGCAGTATCCACATTAGGGAATGGTGTGCGGGTCCTAGCCACTGCAGTGAGAGAGCTAAAAGAATTTGTGAGCAAAAACCTGACTAGTGCAATCAACAGGAACAAATGTGACATTGCTGATCTGAAGATGGCTGTCAGCTTCAGTCAATTCAACAGAAGATTTCTAAATGTTGTGCGGCAGTTTTCAGACAATGCAGGGATAACACCAGCAATATCATTGGACCTGATGACTGATGCTGAGTTGGCCAGAGCTGTATCATACATGCCAACATCTGCAGGGCAGATAAAACTGATGTTGGAGAACCGCGCAATGGTAAGGAGAAAAGGATTTGGAATCCTGATAGGGGTCTACGGAAGCTCTGTGATTTACATGGTTCAATTGCCGATCTTTGGTGTCATAGATACACCTTGTTGGATCATCAAGGCAGCTCCCTCTTGCTCAGAAAAAAACGGGAATTATGCTTGCCTCCTAAGAGAGGATCAAGGGTGGTATTGTAAAAATGCAGGATCTACTGTTTACTACCCAAATGAAAAAGACTGCGAAACAAGAGGTGATCATGTTTTTTGTGACACAGCAGCAGGGATCAATGTTGCTGAGCAATCAAGAGAATGCAACATCAACATATCTACTACCAACTACCCATGCAAAGTCAGCACAGGAAGACACCCTATAAGCATGGTTGCACTATCACCTCTCGGTGCTTTGGTGGCTTGCTATAAAGGGGTAAGCTGCTCGATTGGCA GCAATTGGGTTGGAATCATCAAACAATTACCCAAAGGCTGCTCATACATAACCAACCAGGATGCAGACACTGTAACAATTGACAATACCGTGTATCAACTAAGCAAAGTTGAAGGTGAACAGCATGTAATAAAAGGGAGACCAGTTTCAAGCAGTTTTGATCCAATCAAGTTTCCTGAGGATCAGTTCAATGTTGCGCTTGATCAAGTCTTCGAAAGCATTGAGAACAGTCAGGCACTAGTGGACCAGTCAAACAAAATTCTAAACAGTGCAGAAAAAGGAAACACTGGTTTCATTATCGTAGTAATTTTGGTTGCTGTTCTTGGTCTAACCATGATTTCAGTGAGCATCATCATCATAATCAAGAAAACAAGGAAGCCCACAGGAGCACCTCCAGAGCTGAATGGTGTCACCAACGGCGGTTTCATACCACATAGTTA gb|KJ627414.1|: ATGTCTTGGAAAGTGATGATTATCATTTCGTTACTCATAA 3 3015-4634 HumanCACCTCAGCATGGACTAAAAGAAAGTTATTTAGAAGAAT metapneumo virusCATGTAGTACTATAACTGAAGGATATCTCAGTGTTTTAAG strain hMPV/HomoAACAGGTTGGTACACCAATGTCTTTACATTAGAAGTTGGT sapiens/PER/GATGTTGAAAATCTTACATGTACTGATGGACCTAGCTTAA CFI0497/2010/B,TCAAAACAGAACTTGACCTAACCAAAAGTGCTTTAAGAG complete genomeAACTCAAAACAGTTTCTGCTGATCAGTTAGCGAGAGAAGAACAAATTGAAAATCCCAGACAATCAAGGTTTGTCCTAGGTGCAATAGCTCTTGGAGTTGCCACAGCAGCAGCAGTCACAGCAGGCATTGCAATAGCCAAAACTATAAGGCTTGAGAGTGAAGTGAATGCAATCAAAGGTGCTCTCAAAACAACCAATGAGGCAGTATCAACACTAGGAAATGGAGTGCGGGTCCTAGCCACTGCAGTAAGAGAGCTGAAAGAATTTGTGAGCAAAAACCTGACTAGTGCGATCAACAAGAACAAGTGTGACATTGCTGATTTGAAGATGGCTGTCAGCTTCAGTCAGTTCAACAGAAGATTCCTAAATGTTGTGCGGCAGTTTTCAGACAATGCAGGGATAACACCAGCAATATCATTGGACCTGATGAATGATGCTGAGCTGGCCAGAGCTGTATCATACATGCCAACATCTGCAGGACAGATAAAACTAATGTTAGAGAACCGTGCAATGGTGAGGAGAAAAGGATTTGGAATCTTGATAGGGGTCTACGGAAGCTCTGTGATTTACATGGTCCAGCTGCCGATCTTTGGTGTCATAAATACACCTTGTTGGATAATCAAGGCAGCTCCCTCTTGTTCAGAAAAAGATGGAAATTATGCTTGCCTCCTAAGAGAGGATCAAGGGTGGTATTGTAAAAATGCAGGATCCACTGTTTACTACCCAAATGAAAAAGACTGCGAAACAAGAGGTGATCATGTTTTTTGTGACACAGCAGCAGGGATCAATGTTGCTGAGCAATCAAGAGAATGCAACATCAACATATCTACCACCAACTACCCATGCAAAGTCAGCACAGGAAGACACCCTATCAGCATGGTTGCACTATCACCTCTCGGTGCTTTGGTAGCTTGCTACAAAGGGGTTAGCTGCTCGACTGGCAGTAATCAGGTTGGAATAATCAAACAACTACCTAAAGGCTGCTCATACATAACTAACCAGGACGCAGACACTGTAACAATTGACAACACTGTGTATCAACTAAGCAAAGTTGAGGGTGAACAGCATGTAATAAAAGGGAGACCAGTTTCAAGCAGTTTTGATCCAATCAGGTTTCCTGAGGATCAGTTCAATGTTGCGCTTGATCAAGTCTTTGAAAGCATTGAAAACAGTCAAGCACTAGTGGACCAGTCAAACAAAATTCTGAACAGTGCAGA AAAAGGAAACACTGGTTTCATTATTGTAATAATTTTGATTGCTGTTCTTGGGTTAACCATGATTTCAGTGAGCATCATCATCATAATCAAAAAAACAAGGAAGCCCACAGGGGCACCTCCGGAGCTGAATGGTGT TACCAACGGCGGTTTCATACCGCATAGTTAGgb|KJ723483.1|: ATGGAGTTGCCAATCCTCAAAACAAATGCAATTACCACA 45586-7310 Human ATCCTTGCTGCAGTCACACTCTGTTTCGCTTCCAGTCAAA respiratoryACATCACTGAAGAATTTTATCAATCAACATGCAGTGCAG syncytial virusTTAGCAAAGGCTATCTTAGTGCTCTAAGAACTGGTTGGTA strain RSV A/HomoTACTAGTGTTATAACTATAGAATTAAGTAATATCAAGGA sapiens/USA/84I-AAATAAGTGTAATGGAACAGATGCTAAGGTAAAATTGAT 215A-01/1984,AAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGA complete genomeATTGCAGTTGCTCATGCAAAGCACACCAGCAGCCAACAATCGAGCCAGAAGAGAACTACCAAGGTTTATGAATTATACACTCAATAATACCAAAAATACCAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGCTTTTTGTTAGGTGTTGGATCTGCAATCGCCAGTGGCATTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAAATCAAAAGTGCTCTACTATCCACAAACAAGGCTGTAGTCAGCTTATCAAATGGAGTTAGTGTCTTAACCAGCAAAGTGTTAGACCTCAAAAACTATATAGATAAACAGTTGTTACCTATTGTGAACAAGCAAAGCTGCAGCATATCAAACATTGAAACTGTGATAGAGTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAATTTAGTGTTAATGCAGGTGTAACTACACCTGTAAGCACTTATATGTTAACTAATAGTGAATTATTATCATTAATCAATGATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAGCAAAGTTACTCTATCATGTCCATAATAAAGGAGGAAGTCTTAGCATATGTAGTACAATTACCACTATATGGTGTAATAGATACACCCTGTTGGAAACTGCACACATCCCCTCTATGTACAACCAACACAAAGGAAGGGTCCAACATCTGCTTAACAAGAACCGACAGAGGATGGTATTGTGACAATGCAGGATCAGTATCTTTCTTCCCACAAGCTGAAACATGTAAAGTTCAATCGAATCGGGTATTTTGTGACACAATGAACAGTTTAACATTACCAAGTGAAGTAAATCTCTGCAACATTGACATATTCAACCCCAAATATGATTGCAAAATTATGACTTCAAAAACAGATGTAAGCAGCTCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAATAAAAATCGTGGGATCATAAAGACATTTTCTAACGGGTGTGATTATGTATCAAATAAGGGGGTGGATACTGTGTCTGTAGGTAATACATTATATTATGTAAATAAGCAAGAAGGCAAAAGTCTCTATGTAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTGTTCCCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAATGAGAAGATTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAAATGCTGGTAAATCCACCACAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCAGTTGGACTGCTCCTATACTGCAAGGCCAGAAGCACACCAGTCACACTAAGTAAGGATCAACTGA GTGGTATAAATAATATTGCATTTAGTAACTGAhMPV mRNA Sequences gi|122891979|gb|AUGAGCUGGAAGGUGGUGAUUAUCUUCAGCCUGCUGAU 57 EF051124.11 HumanUACACCUCAACACGGCCUGAAGGAGAGCUACCUGGAAG metapneumo virusAGAGCUGCUCCACCAUCACCGAGGGCUACCUGAGCGUG isolate TN/92-4CUGCGGACCGGCUGGUACACCAACGUGUUCACCCUGGA fusion protein gene,GGUGGGCGACGUGGAGAACCUGACCUGCAGCGACGGCC complete genomeCUAGCCUGAUCAAGACCGAGCUGGACCUGACCAAGAGCGCUCUGAGAGAGCUGAAGACCGUGUCCGCCGACCAGCUGGCCAGAGAGGAACAGAUCGAGAACCCUCGGCAGAGCAGAUUCGUGCUGGGCGCCAUCGCUCUGGGAGUCGCCGCUGCCGCUGCAGUGACAGCUGGAGUGGCCAUUGCUAAGACCAUCAGACUGGAAAGCGAGGUGACAGCCAUCAACAAUGCCCUGAAGAAGACCAACGAGGCCGUGAGCACCCUGGGCAAUGGAGUGAGAGUGCUGGCCACAGCCGUGCGGGAGCUGAAGGACUUCGUGAGCAAGAACCUGACCAGAGCCAUCAACAAGAACAAGUGCGACAUCGAUGACCUGAAGAUGGCCGUGAGCUUCUCCCAGUUCAACAGACGGUUCCUGAACGUGGUGAGACAGUUCUCCGACAACGCUGGAAUCACACCUGCCAUUAGCCUGGACCUGAUGACCGACGCCGAGCUGGCUAGAGCCGUGCCCAACAUGCCCACCAGCGCUGGCCAGAUCAAGCUGAUGCUGGAGAACAGAGCCAUGGUGCGGAGAAAGGGCUUCGGCAUCCUGAUUGGGGUGUAUGGAAGCUCCGUGAUCUACAUGGUGCAGCUGCCCAUCUUCGGCGUGAUCGACACACCCUGCUGGAUCGUGAAGGCCGCUCCUAGCUGCUCCGAGAAGAAAGGAAACUAUGCCUGUCUGCUGAGAGAGGACCAGGGCUGGUACUGCCAGAACGCCGGAAGCACAGUGUACUAUCCCAACGAGAAGGACUGCGAGACCAGAGGCGACCACGUGUUCUGCGACACCGCUGCCGGAAUCAACGUGGCCGAGCAGAGCAAGGAGUGCAACAUCAACAUCAGCACAACCAACUACCCCUGCAAGGUGAGCACCGGACGGCACCCCAUCAGCAUGGUGGCUCUGAGCCCUCUGGGCGCUCUGGUGGCCUGCUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAAUCGGGUGGGCAUCAUCAAGCAGCUGAACAAGGGAUGCUCCUACAUCACCAACCAGGACGCCGACACCGUGACCAUCGACAACACCGUGUACCAGCUGAGCAAGGUGGAGGGCGAGCAGCACGUGAUCAAGGGCAGACCCGUGAGCUCCAGCUUCGACCCCAUCAAGUUCCCUGAGGACCAGUUCAACGUGGCCCUGGACCAGGUGUUUGAGAACAUCGAGAACAGCCAGGCCCUGGUGGACCAGAGCAACAGAAUCCUGUCCAGCGCUGAGAAGGGCAACACCGGCUUCAUCAUUGUGAUCAUUCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGAGCAUCUUCAUCAUUAUCAAGAAGACCAAGAAACCCACCGGAGCCCCUCCUGAGCUGAGCGGCGUGACCAAC AAUGGCUUCAUUCCCCACAACUGAgb|AY525843.1|:  AUGUCUUGGAAAGUGAUGAUCAUCAUUUCGUUACUCAU 583065-4684 Human AACACCCCAGCACGGGCUAAAGGAGAGUUAUUUGGAAG metapneumo virusAAUCAUGUAGUACUAUAACUGAGGGAUACCUCAGUGUU isolate NL/1/99,UUAAGAACAGGCUGGUACACUAAUGUCUUCACAUUAGA complete genomeAGUUGGUGAUGUUGAAAAUCUUACAUGUACUGAUGGACCUAGCUUAAUCAAAACAGAACUUGAUCUAACAAAAAGUGCUUUAAGGGAACUCAAAACAGUCUCUGCUGAUCAGUUGGCGAGAGAGGAGCAAAUUGAAAAUCCCAGACAAUCAAGAUUUGUCUUAGGUGCGAUAGCUCUCGGAGUUGCUACAGCAGCAGCAGUCACAGCAGGCAUUGCAAUAGCCAAAACCAUAAGGCUUGAGAGUGAGGUGAAUGCAAUUAAAGGUGCUCUCAAACAAACUAAUGAAGCAGUAUCCACAUUAGGGAAUGGUGUGCGGGUCCUAGCCACUGCAGUGAGAGAGCUAAAAGAAUUUGUGAGCAAAAACCUGACUAGUGCAAUCAACAGGAACAAAUGUGACAUUGCUGAUCUGAAGAUGGCUGUCAGCUUCAGUCAAUUCAACAGAAGAUUUCUAAAUGUUGUGCGGCAGUUUUCAGACAAUGCAGGGAUAACACCAGCAAUAUCAUUGGACCUGAUGACUGAUGCUGAGUUGGCCAGAGCUGUAUCAUACAUGCCAACAUCUGCAGGGCAGAUAAAACUGAUGUUGGAGAACCGCGCAAUGGUAAGGAGAAAAGGAUUUGGAAUCCUGAUAGGGGUCUACGGAAGCUCUGUGAUUUACAUGGUUCAAUUGCCGAUCUUUGGUGUCAUAGAUACACCUUGUUGGAUCAUCAAGGCAGCUCCCUCUUGCUCAGAAAAAAACGGGAAUUAUGCUUGCCUCCUAAGAGAGGAUCAAGGGUGGUAUUGUAAAAAUGCAGGAUCUACUGUUUACUACCCAAAUGAAAAAGACUGCGAAACAAGAGGUGAUCAUGUUUUUUGUGACACAGCAGCAGGGAUCAAUGUUGCUGAGCAAUCAAGAGAAUGCAACAUCAACAUAUCUACUACCAACUACCCAUGCAAAGUCAGCACAGGAAGACACCCUAUAAGCAUGGUUGCACUAUCACCUCUCGGUGCUUUGGUGGCUUGCUAUAAAGGGGUAAGCUGCUCGAU UGGCAGCAAUUGGGUUGGAAUCAUCAAACAAUUACCCAAAGGCUGCUCAUACAUAACCAACCAGGAUGCAGACACUGUAACAAUUGACAAUACCGUGUAUCAACUAAGCAAAGUUGAAGGUGAACAGCAUGUAAUAAAAGGGAGACCAGUUUCAAGCAGUUUUGAUCCAAUCAAGUUUCCUGAGGAUCAGUUCAAUGUUGCGCUUGAUCAAGUCUUCGAAAGCAUUGAGAACAGUCAGGCACUAGUGGACCAGUCAAACAAAAUUCUAAACAGUGCAGAAAAAGGAAACACUGGUUUCAUUAUCGUAGUAAUUUUGGUUGCUGUUCUUGGUCUAACCAUGAUUUCAGUGAGCAUCAUCAUCAUAAUCAAGAAAACAAGGAAGCCCACAGGAGCACCUCCAGAGCUGAAUGGUGUCACCAACGGCGGUUUCAU ACCACAUAGUUAG gb|KJ627414.1|:AUGUCUUGGAAAGUGAUGAUUAUCAUUUCGUUACUCAU 59 3015-4634 Human AACACCUCAGCAUGGACUAAAAGAAAGUUAUUUAGAAG metapneumo virusAAUCAUGUAGUACUAUAACUGAAGGAUAUCUCAGUGUU strain hMPV/HomoUUAAGAACAGGUUGGUACACCAAUGUCUUUACAUUAGA sapiens/PER/AGUUGGUGAUGUUGAAAAUCUUACAUGUACUGAUGGA CFI0497/2010/B,CCUAGCUUAAUCAAAACAGAACUUGACCUAACCAAAAG complete genomeUGCUUUAAGAGAACUCAAAACAGUUUCUGCUGAUCAGUUAGCGAGAGAAGAACAAAUUGAAAAUCCCAGACAAUCAAGGUUUGUCCUAGGUGCAAUAGCUCUUGGAGUUGCCACAGCAGCAGCAGUCACAGCAGGCAUUGCAAUAGCCAAAACUAUAAGGCUUGAGAGUGAAGUGAAUGCAAUCAAAGGUGCUCUCAAAACAACCAAUGAGGCAGUAUCAACACUAGGAAAUGGAGUGCGGGUCCUAGCCACUGCAGUAAGAGAGCUGAAAGAAUUUGUGAGCAAAAACCUGACUAGUGCGAUCAACAAGAACAAGUGUGACAUUGCUGAUUUGAAGAUGGCUGUCAGCUUCAGUCAGUUCAACAGAAGAUUCCUAAAUGUUGUGCGGCAGUUUUCAGACAAUGCAGGGAUAACACCAGCAAUAUCAUUGGACCUGAUGAAUGAUGCUGAGCUGGCCAGAGCUGUAUCAUACAUGCCAACAUCUGCAGGACAGAUAAAACUAAUGUUAGAGAACCGUGCAAUGGUGAGGAGAAAAGGAUUUGGAAUCUUGAUAGGGGUCUACGGAAGCUCUGUGAUUUACAUGGUCCAGCUGCCGAUCUUUGGUGUCAUAAAUACACCUUGUUGGAUAAUCAAGGCAGCUCCCUCUUGUUCAGAAAAAGAUGGAAAUUAUGCUUGCCUCCUAAGAGAGGAUCAAGGGUGGUAUUGUAAAAAUGCAGGAUCCACUGUUUACUACCCAAAUGAAAAAGACUGCGAAACAAGAGGUGAUCAUGUUUUUUGUGACACAGCAGCAGGGAUCAAUGUUGCUGAGCAAUCAAGAGAAUGCAACAUCAACAUAUCUACCACCAACUACCCAUGCAAAGUCAGCACAGGAAGACACCCUAUCAGCAUGGUUGCACUAUCACCUCUCGGUGCUUUGGUAGCUUGCUACAAAGGGGUUAGCUGCUCGACUGGCAGUAAUCAGGUUGGAAUAAUCAAACAACUACCUAAAGGCUGCUCAUACAUAACUAACCAGGACGCAGACACUGUAACAAUUGACAACACUGUGUAUCAACUAAGCAAAGUUGAGGGUGAACAGCAUGUAAUAAAAGGGAGACCAGUUUCAAGCAGUUUUGAUCCAAUCAGGUUUCCUGAGGAUCAGUUCAAUGUUGCGCUUGAUCAAGUCUUUGAAAGCAUUGAAAACAGUCAAGCACUAGUGGACCAGUCAAACAAAA UUCUGAACAGUGCAGAAAAAGGAAACACUGGUUUCAUUAUUGUAAUAAUUUUGAUUGCUGUUCUUGGGUUAACCAUGAUUUCAGUGAGCAUCAUCAUCAUAAUCAAAAAAACAAGGAAGCCCACAGGGGCACCUCCGGAGCUGAAUGGUGUUACCAACGGCGGUUUCAUACCGCAUAGUUAG gb|KJ723483.1|:AUGGAGUUGCCAAUCCUCAAAACAAAUGCAAUUACCAC 60 5586-7310 HumanAAUCCUUGCUGCAGUCACACUCUGUUUCGCUUCCAGUC respiratoryAAAACAUCACUGAAGAAUUUUAUCAAUCAACAUGCAGU syncytial virusGCAGUUAGCAAAGGCUAUCUUAGUGCUCUAAGAACUGG strain RSVA/HomoUUGGUAUACUAGUGUUAUAACUAUAGAAUUAAGUAAU sapiens/USA/84I-AUCAAGGAAAAUAAGUGUAAUGGAACAGAUGCUAAGG 215A-01/1984,UAAAAUUGAUAAAACAAGAAUUAGAUAAAUAUAAAAA complete genomeUGCUGUAACAGAAUUGCAGUUGCUCAUGCAAAGCACACCAGCAGCCAACAAUCGAGCCAGAAGAGAACUACCAAGGUUUAUGAAUUAUACACUCAAUAAUACCAAAAAUACCAAUGUAACAUUAAGCAAGAAAAGGAAAAGAAGAUUUCUUGGCUUUUUGUUAGGUGUUGGAUCUGCAAUCGCCAGUGGCAUUGCUGUAUCUAAGGUCCUGCACCUAGAAGGGGAAGUGAACAAAAUCAAAAGUGCUCUACUAUCCACAAACAAGGCUGUAGUCAGCUUAUCAAAUGGAGUUAGUGUCUUAACCAGCAAAGUGUUAGACCUCAAAAACUAUAUAGAUAAACAGUUGUUACCUAUUGUGAACAAGCAAAGCUGCAGCAUAUCAAACAUUGAAACUGUGAUAGAGUUCCAACAAAAGAACAACAGACUACUAGAGAUUACCAGGGAAUUUAGUGUUAAUGCAGGUGUAACUACACCUGUAAGCACUUAUAUGUUAACUAAUAGUGAAUUAUUAUCAUUAAUCAAUGAUAUGCCUAUAACAAAUGAUCAGAAAAAGUUAAUGUCCAACAAUGUUCAAAUAGUUAGACAGCAAAGUUACUCUAUCAUGUCCAUAAUAAAGGAGGAAGUCUUAGCAUAUGUAGUACAAUUACCACUAUAUGGUGUAAUAGAUACACCCUGUUGGAAACUGCACACAUCCCCUCUAUGUACAACCAACACAAAGGAAGGGUCCAACAUCUGCUUAACAAGAACCGACAGAGGAUGGUAUUGUGACAAUGCAGGAUCAGUAUCUUUCUUCCCACAAGCUGAAACAUGUAAAGUUCAAUCGAAUCGGGUAUUUUGUGACACAAUGAACAGUUUAACAUUACCAAGUGAAGUAAAUCUCUGCAACAUUGACAUAUUCAACCCCAAAUAUGAUUGCAAAAUUAUGACUUCAAAAACAGAUGUAAGCAGCUCCGUUAUCACAUCUCUAGGAGCCAUUGUGUCAUGCUAUGGCAAAACUAAAUGUACAGCAUCCAAUAAAAAUCGUGGGAUCAUAAAGACAUUUUCUAACGGGUGUGAUUAUGUAUCAAAUAAGGGGGUGGAUACUGUGUCUGUAGGUAAUACAUUAUAUUAUGUAAAUAAGCAAGAAGGCAAAAGUCUCUAUGUAAAAGGUGAACCAAUAAUAAAUUUCUAUGACCCAUUAGUGUUCCCCUCUGAUGAAUUUGAUGCAUCAAUAUCUCAAGUCAAUGAGAAGAUUAACCAGAGCCUAGCAUUUAUUCGUAAAUCCGAUGAAUUAUUACAUAAUGUAAAUGCUGGUAAAUCCACCACAAAUAUCAUGAUAACUACUAUAAUUAUAGUGAUUAUAGUAAUAUUGUUAUCAUUAAUUGCAGUUGGACUGCUCCUAUACUGCAAGGCCAGAAGCACACCAGUCACACUAAGUAAGGAUCAACUGAGUGGUAU AAAUAAUAUUGCAUUUAGUAACUGA

TABLE 3  hMPV Amino Acid Sequences SEQ ID Description Sequence NO:gi|122891979|gb| MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGW 5EF051124.1| Human  YTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSmetapneumo virus  ADQLAREEQIENPRQSRFVLGAIALGVAAAAAVTAGVAIAKisolate TN/92-4 TIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDfusion protein gene, FVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFScomplete cds DNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN gb|AY525843.1|: MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGW 6 3065-4684 HumanYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVS metapneumo virus ADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKT isolate NL/1/99, IRLESEVNAIKGALKQTNEAVSTLGNGVRVLATAVRELKEF complete cdsVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTGFIIVVILVAVLGLTMISVSIIIIIKKTRKPTGAPPELNGVTNGGFIPHS gb|KJ627414.1|: MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGW 7 3015-4634 HumanYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVS metapneumo virus ADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKT strain hMPV/Homo IRLESEVNAIKGALKTTNEAVSTLGNGVRVLATAVRELKEF sapiens/PER/CFI04VSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSD 97/2010/B,NAGITPAISLDLMNDAELARAVSYMPTSAGQIKLMLENRAM complete cdsVRRKGFGILIGVYGSSVIYMVQLPIFGVINTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSTGSNQVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIRFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTGFIIVIILIAVLGLTMISVSIIIIIKKTRKPTGAPPELNGVTNGGFIPHS gb|KJ723483.1|: MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKG  8 5586-7310 HumanYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDK respiratoryYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNT syncytial virusNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKI strain RSVA/HomoKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVN sapiens/USA/84I-KQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYM 215A-01/1984,LTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKE complete cdsEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLL YCKARSTPVTLSKDQLSGINNIAFSN

TABLE 4 hMPV NCBI Accession Numbers (Amino Acid Sequences) Virus GenBankAccession F [Human metapneumovirus] [Human metapneumovirus] AEK26895.1fusion glycoprotein [Human metapneumovirus] ACJ53565.1 fusionglycoprotein [Human metapneumovirus] ACJ53566.1 fusion glycoprotein[Human metapneumovirus] ACJ53569.1 fusion protein [Humanmetapneumovirus] AEZ52347.1 fusion glycoprotein [Human metapneumovirus]ACJ53574.1 fusion glycoprotein [Human metapneumovirus] AHV79473.1 fusionglycoprotein [Human metapneumovirus] ACJ53570.1 fusion glycoprotein[Human metapneumovirus] ACJ53567.1 fusion protein [Humanmetapneumovirus] AAS22125.1 fusion glycoprotein [Human metapneumovirus]AHV79795.1 fusion glycoprotein [Human metapneumovirus] AHV79455.1 fusionglycoprotein [Human metapneumovirus] ACJ53568.1 fusion protein [Humanmetapneumovirus] AAS22109.1 fusion glycoprotein [Human metapneumovirus]AGU68417.1 fusion glycoprotein [Human metapneumovirus] AGJ74228.1 fusionglycoprotein [Human metapneumovirus] ACJ53575.1 fusion protein [Humanmetapneumovirus] AAU25820.1 fusion glycoprotein [Human metapneumovirus]AGU68377.1 fusion glycoprotein [Human metapneumovirus] AGU68371.1 fusionglycoprotein [Human metapneumovirus] AGJ74087.1 fusion glycoprotein[Human metapneumovirus] ACJ53560.1 fusion glycoprotein [Humanmetapneumovirus] AHV79858.1 fusion glycoprotein [Human metapneumovirus]ACJ53577.1 fusion protein [Human metapneumovirus] AAS22085.1 fusionprotein [Human metapneumovirus] AEZ52348.1 fusion glycoprotein [Humanmetapneumovirus] AGJ74044.1 fusion glycoprotein [Human metapneumovirus]ACJ53563.1 fusion glycoprotein precursor [Human metapneumovirus]YP_012608.1 fusion glycoprotein [Human metapneumovirus] AGJ74053.1fusion protein [Human metapneumovirus] BAM37562.1 fusion glycoprotein[Human metapneumovirus] ACJ53561.1 fusion glycoprotein [Humanmetapneumovirus] AGU68387.1 fusion [Human metapneumovirus] AGL74060.1fusion glycoprotein precursor [Human metapneumovirus] AAV88364.1 fusionprotein [Human metapneumovirus] AAN52910.1 fusion protein [Humanmetapneumovirus] AAN52915.1 fusion protein [Human metapneumovirus]BAM37564.1 fusion glycoprotein precursor [Human metapneumovirus]BAH59618.1 fusion protein [Human metapneumovirus] AAQ90144.1 fusionglycoprotein [Human metapneumovirus] AHV79446.1 fusion protein [Humanmetapneumovirus] AEL87260.1 fusion glycoprotein [Human metapneumovirus]AHV79867.1 fusion protein [Human metapneumovirus] ABQ66027.2 fusionglycoprotein [Human metapneumovirus] ACJ53621.1 fusion protein [Humanmetapneumovirus] AAN52911.1 fusion glycoprotein [Human metapneumovirus]AHV79536.1 fusion glycoprotein [Human metapneumovirus] AGU68411.1 fusionprotein [Human metapneumovirus] AEZ52346.1 fusion protein [Humanmetapneumovirus] AAN52913.1 fusion protein [Human metapneumovirus]AAN52908.1 fusion glycoprotein [Human metapneumovirus] ACJ53553.1 fusionglycoprotein [Human metapneumovirus] AIY25727.1 fusion protein [Humanmetapneumovirus] ABM67072.1 fusion protein [Human metapneumovirus]AEZ52361.1 fusion protein [Human metapneumovirus] AAS22093.1 fusionglycoprotein [Human metapneumovirus] AGH27049.1 fusion protein [Humanmetapneumovirus] AAK62968.2 fusion glycoprotein [Human metapneumovirus]ACJ53556.1 fusion glycoprotein [Human metapneumovirus] ACJ53620.1 fusionprotein [Human metapneumovirus] ABQ58820.1 F [Human metapneumovirus][Human metapneumovirus] AEK26886.1 fusion glycoprotein [Humanmetapneumovirus] ACJ53619.1 fusion glycoprotein [Human metapneumovirus]ACJ53555.1 fusion [Human metapneumovirus] AGL74057.1 fusion protein[Human metapneumovirus] ABD27850.1 fusion protein [Humanmetapneumovirus] AEZ52349.1 fusion protein [Human metapneumovirus]ABD27848.1 fusion protein [Human metapneumovirus] ABD27846.1 fusionprotein [Human metapneumovirus] ABQ66021.1 fusion protein [Humanmetapneumovirus] AFM57710.1 fusion protein [Human metapneumovirus]AFM57709.1 fusion protein [Human metapneumovirus] ABH05968.1 fusionprotein [Human metapneumovirus] AEZ52350.1 fusion protein [Humanmetapneumovirus] AFM57712.1 fusion protein [Human metapneumovirus]AEZ52364.1 fusion protein [Human metapneumovirus] AAN52912.1 fusionprotein [Human metapneumovirus] AEZ52363.1 fusion [Humanmetapneumovirus] AGL74059.1 fusion glycoprotein [Human metapneumovirus]ACJ53583.1 fusion protein [Human metapneumovirus] AEZ52356.1 fusionprotein [Human metapneumovirus] AEZ52353.1 fusion glycoprotein [Humanmetapneumovirus] ACJ53581.1 fusion glycoprotein [Human metapneumovirus]ACJ53578.1 fusion protein [Human metapneumovirus] AAS22117.1 fusionprotein [Human metapneumovirus] BAN75965.1 fusion protein [Humanmetapneumovirus] AGF92105.1 fusion protein [Human metapneumovirus]AAS22077.1 fusion protein [Human metapneumovirus] AAN52909.1 fusionglycoprotein [Human metapneumovirus] ACJ53586.1 fusion protein [Humanmetapneumovirus] AAQ90145.1 fusion glycoprotein [Human metapneumovirus]AGT75042.1 fusion [Human metapneumovirus] AGL74058.1 fusion protein[Human metapneumovirus] AEL87263.1 fusion glycoprotein [Humanmetapneumovirus] AGH27057.1 fusion glycoprotein [Human metapneumovirus]AHV79491.1 F [Human metapneumovirus] [Human metapneumovirus] AEK26906.1fusion glycoprotein [Human metapneumovirus] ACJ53580.1 fusion protein[Human metapneumovirus] AEZ52354.1 fusion protein [Humanmetapneumovirus] AAN52914.1 G [Human metapneumovirus] [Humanmetapneumovirus] AEK26901.1 glycoprotein [Human metapneumovirus]AFI56738.1 glycoprotein [Human metapneumovirus] AFI56739.1 glycoprotein[Human metapneumovirus] AFI56745.1 G protein [Human metapneumovirus]AAQ62718.1 G protein [Human metapneumovirus] AAQ62719.1 attachmentglycoprotein G [Human metapneumovirus] AGH27104.1 G protein [Humanmetapneumovirus] AAQ62729.1 G protein [Human metapneumovirus] AAQ62728.1glycoprotein [Human metapneumovirus] AFI56753.1 glycoprotein [Humanmetapneumovirus] AFI56746.1 glycoprotein [Human metapneumovirus]AFI56750.1 glycoprotein [Human metapneumovirus] AFI56747.1 G protein[Human metapneumovirus] AAQ62721.1 glycoprotein [Human metapneumovirus]AAT46573.1 glycoprotein [Human metapneumovirus] AFI56748.1 glycoprotein[Human metapneumovirus] AFI56736.1 glycoprotein [Human metapneumovirus]AFI56749.1 attachment glycoprotein G [Human metapneumovirus] AGH27131.1attachment glycoprotein G [Human metapneumovirus] AHV79558.1glycoprotein [Human metapneumovirus] AFI56740.1 glycoprotein [Humanmetapneumovirus] AFI56741.1 glycoprotein [Human metapneumovirus]AFI56744.1 attachment glycoprotein G [Human metapneumovirus] AHV79790.1attachment glycoprotein G [Human metapneumovirus] AGH27122.1 attachmentglycoprotein G [Human metapneumovirus] AHV79763.1 attachmentglycoprotein G [Human metapneumovirus] AGZ48849.1 glycoprotein [Humanmetapneumovirus] AFI56743.1 attachment glycoprotein G [Humanmetapneumovirus] AHV79450.1 glycoprotein [Human metapneumovirus]AFI56751.1 attachment glycoprotein [Human metapneumovirus] AAS48482.1attachment glycoprotein G [Human metapneumovirus] AHV79889.1 attachmentsurface glycoprotein [Human metapneumovirus] AGW43050.1 glycoprotein[Human metapneumovirus] AFI56754.1 attachment glycoprotein G [Humanmetapneumovirus] AHV79601.1 glycoprotein [Human metapneumovirus]AFI56752.1 attachment glycoprotein G [Human metapneumovirus] AHV79871.1G protein [Human metapneumovirus] AEZ68099.1 attachment glycoprotein G[Human metapneumovirus] AHV79817.1 attachment glycoprotein G [Humanmetapneumovirus] AHV79943.1 attachment glycoprotein G [Humanmetapneumovirus] BAN75968.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43045.1 attachment glycoprotein G [Humanmetapneumovirus] AHV79628.1 attachment glycoprotein [Humanmetapneumovirus] AFK49783.1 G protein [Human metapneumovirus] AAQ62723.1attachment glycoprotein [Human metapneumovirus] ABD27839.1 attachmentsurface glycoprotein [Human metapneumovirus] AGW43046.1 G protein [Humanmetapneumovirus] AAQ62717.1 glycoprotein [Human metapneumovirus]AFI56742.1 attachment protein [Human metapneumovirus] ABQ44522.1glycoprotein [Human metapneumovirus] AFI56735.1 attachment surfaceglycoprotein [Human metapneumovirus] AGW43065.1 G protein [Humanmetapneumovirus] AAQ62724.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43075.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43062.1 glycoprotein [Human metapneumovirus]AAT46579.1 attachment surface glycoprotein [Human metapneumovirus]AGW43064.1 attachment surface glycoprotein [Human metapneumovirus]AGW43054.1 attachment surface glycoprotein [Human metapneumovirus]AGW43042.1 attachment surface glycoprotein [Human metapneumovirus]AGW43078.1 attachment surface glycoprotein [Human metapneumovirus]AGW43067.1 G protein [Human metapneumovirus] AAQ62722.1 attachmentsurface glycoprotein [Human metapneumovirus] AGW43063.1 glycoprotein[Human metapneumovirus] AAT46571.1 glycoprotein [Human metapneumovirus]AAT46578.1 attachment glycoprotein G [Human metapneumovirus] AGJ74232.1glycoprotein [Human metapneumovirus] AAT46580.1 glycoprotein [Humanmetapneumovirus] AAT46574.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43061.1 attachment glycoprotein [Humanmetapneumovirus] AFK49791.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43047.1 glycoprotein [Human metapneumovirus]ABC26386.1 attachment glycoprotein [Human metapneumovirus] AAS48466.1attachment surface glycoprotein [Human metapneumovirus] AGW43048.1attachment glycoprotein G [Human metapneumovirus] AGH27140.1 attachmentsurface glycoprotein [Human metapneumovirus] AGW43049.1 attachmentglycoprotein G [Human metapneumovirus] AGJ74082.1 attachmentglycoprotein G [Human metapneumovirus] AHV79442.1 attachmentglycoprotein G [Human metapneumovirus] AGJ74091.1 attachmentglycoprotein G [Human metapneumovirus] AHV79477.1 attachment surfaceglycoprotein [Human metapneumovirus] AGW43056.1 attachment protein[Human metapneumovirus] ABQ44523.1 attachment glycoprotein G [Humanmetapneumovirus] BAH59622.1 attachment surface glycoprotein [Humanmetapneumovirus] AGW43070.1 glycoprotein [Human metapneumovirus]AAT46585.1 attachment glycoprotein G [Human metapneumovirus] AGU68409.1attachment glycoprotein G [Human metapneumovirus] AGJ74223.1 attachmentglycoprotein [Human metapneumovirus] AAS22129.1 attachment glycoproteinG [Human metapneumovirus] AGJ74048.1 G protein [Human metapneumovirus]AAQ62725.1 glycoprotein [Human metapneumovirus] ABC26384.1 attachmentprotein [Human metapneumovirus] ABQ44525.1 attachment glycoprotein G[Human metapneumovirus] YP_012612.1 attachment surface glycoprotein[Human metapneumovirus] AGW43071.1 attachment glycoprotein G [Humanmetapneumovirus] AGJ74162.1 attachment glycoprotein G [Humanmetapneumovirus] AGH27095.1 attachment glycoprotein G [Humanmetapneumovirus] AHV79531.1 G protein [Human metapneumovirus] AAQ62726.1attachment glycoprotein [Human metapneumovirus] AAS48465.1 attachmentsurface glycoprotein [Human metapneumovirus] AGW43058.1 P [Humanmetapneumovirus] [Human metapneumovirus] AEK26894.1 phosphoprotein[Human metapneumovirus] AHV79631.1 phosphoprotein [Humanmetapneumovirus] AHV79901.1 phosphoprotein [Human metapneumovirus]AHV79570.1 phosphoprotein [Human metapneumovirus] AGJ74076.1phosphoprotein [Human metapneumovirus] AAS22123.1 phosphoprotein [Humanmetapneumovirus] ABB16895.1 phosphoprotein [Human metapneumovirus]AHV79579.1 phosphoprotein [Human metapneumovirus] AGJ74244.1phosphoprotein [Human metapneumovirus] AHV79856.1 phosphoprotein [Humanmetapneumovirus] ACJ70113.1 phosphoprotein [Human metapneumovirus]AGZ48843.1 phosphoprotein [Human metapneumovirus] AHV79498.1phosphoprotein [Human metapneumovirus] AHV79480.1 phosphoprotein [Humanmetapneumovirus] ABQ43382.1 phosphoprotein [Human metapneumovirus]AAS22107.1 phosphoprotein [Human metapneumovirus] ABB16898.1phosphoprotein [Human metapneumovirus] AGH27134.1 phosphoprotein [Humanmetapneumovirus] ABB16899.1 phosphoprotein [Human metapneumovirus]AGH27098.1 phosphoprotein [Human metapneumovirus] AAN52866.1phosphoprotein [Human metapneumovirus] AAS22083.1 phosphoprotein [Humanmetapneumovirus] YP_012606.1 phosphoprotein [Human metapneumovirus]AHV79973.1 phosphoprotein [Human metapneumovirus] AHV79462.1phosphoprotein [Human metapneumovirus] AGJ74042.1 phosphoprotein [Humanmetapneumovirus] AAV88362.1 P [Human metapneumovirus] [Humanmetapneumovirus] AIL23591.1 phosphoprotein [Human metapneumovirus]AHV79453.1 phosphoprotein [Human metapneumovirus] AGJ74261.1phosphoprotein [Human metapneumovirus] AGH27116.1 phosphoprotein [Humanmetapneumovirus] ABB16444.1 phosphoprotein [Human metapneumovirus]ABB16445.1 phosphoprotein [Human metapneumovirus] AHV79507.1phosphoprotein [Human metapneumovirus] BAH59616.1 phosphoprotein [Humanmetapneumovirus] ABB16443.1 phosphoprotein [Human metapneumovirus]ABQ43388.1 phosphoprotein [Human metapneumovirus] ABQ43389.1phosphoprotein [Human metapneumovirus] ABQ43395.1 phosphoprotein [Humanmetapneumovirus] ABQ43385.1 phosphoprotein [Human metapneumovirus]AAP84042.1 phosphoprotein [Human metapneumovirus] AAN52868.1phosphoprotein [Human metapneumovirus] AAP84041.1 phosphoprotein [Humanmetapneumovirus] AGH27080.1 phosphoprotein [Human metapneumovirus]ABQ43387.1 phosphoprotein [Human metapneumovirus] AAS22099.1phosphoprotein [Human metapneumovirus] ABB16896.1 phosphoprotein [Humanmetapneumovirus] AGJ74094.1 phosphoprotein [Human metapneumovirus]AEZ68089.1 phosphoprotein [Human metapneumovirus] ABK97002.1phosphoprotein [Human metapneumovirus] AAP13486.1 phosphoprotein [Humanmetapneumovirus] AHV79444.1 phosphoprotein [Human metapneumovirus]AHV79865.1 phosphoprotein [Human metapneumovirus] AGJ74226.1phosphoprotein [Human metapneumovirus] ABQ43383.1 phosphoprotein [Humanmetapneumovirus] AAN52863.1 phosphoprotein [Human metapneumovirus]AHV79775.1 phosphoprotein [Human metapneumovirus] AEZ68094.1phosphoprotein [Human metapneumovirus] AHV79883.1 phosphoprotein [Humanmetapneumovirus] AEZ68092.1 phosphoprotein [Human metapneumovirus]ABQ43390.1 phosphoprotein [Human metapneumovirus] ABQ43386.1phosphoprotein [Human metapneumovirus] ABQ43391.1 phosphoprotein [Humanmetapneumovirus] ACS16062.1 phosphoprotein [Human metapneumovirus]AEZ68090.1 phosphoprotein [Human metapneumovirus] AAK62967.1phosphoprotein [Human metapneumovirus] AEZ68093.1 phosphoprotein [Humanmetapneumovirus] AEZ68088.1 phosphoprotein [Human metapneumovirus]ABQ43392.1 phosphoprotein [Human metapneumovirus] ABQ43393.1phosphoprotein [Human metapneumovirus] ABQ43384.1 phosphoprotein [Humanmetapneumovirus] ABQ43394.1 phosphoprotein [Human metapneumovirus]ABK96999.1 phosphoprotein [Human metapneumovirus] AHV79489.1phosphoprotein [Human metapneumovirus] AGJ74235.1 phosphoprotein [Humanmetapneumovirus] AAS22075.1 phosphoprotein [Human metapneumovirus]AAS22115.1 phosphoprotein [Human metapneumovirus] AII17601.1phosphoprotein [Human metapneumovirus] ABK97000.1 phosphoprotein [Humanmetapneumovirus] AHV79561.1 phosphoprotein [Human metapneumovirus]AGT75040.1 phosphoprotein [Human metapneumovirus] AAN52864.1phosphoprotein [Human metapneumovirus] ABK97001.1 phosphoprotein [Humanmetapneumovirus] AGT74979.1 phosphoprotein [Human metapneumovirus]AHV79955.1 phosphoprotein [Human metapneumovirus] AGH27055.1phosphoprotein [Human metapneumovirus] AAV88361.1 phosphoprotein [Humanmetapneumovirus] ABQ43397.1 phosphoprotein [Human metapneumovirus]AGJ74173.1 P [Human metapneumovirus] [Human metapneumovirus] AEK26904.1phosphoprotein [Human metapneumovirus] ACJ70104.1 phosphoprotein [Humanmetapneumovirus] ABK97003.1 phosphoprotein [Human metapneumovirus]AGT74955.1 phosphoprotein [Human metapneumovirus] AAN52856.1phosphoprotein [Human metapneumovirus] AAN52862.1 phosphoprotein [Humanmetapneumovirus] AGJ74138.1 phosphoprotein [Human metapneumovirus]AHV79613.1 phosphoprotein [Human metapneumovirus] AGJ74060.1phosphoprotein [Human metapneumovirus] AAQ67684.1 phosphoprotein [Humanmetapneumovirus] AEA02278.1 N [Human metapneumovirus] [Humanmetapneumovirus] AEK26899.1 nucleoprotein [Human metapneumovirus]ACS16061.1 nucleoprotein [Human metapneumovirus] AAS88425.1nucleoprotein [Human metapneumovirus] YP_012605.1 nucleoprotein [Humanmetapneumovirus] AHV79882.1 nucleoprotein [Human metapneumovirus]AHV79774.1 nucleocapsid protein [Human metapneumovirus] AAN52886.1nucleoprotein [Human metapneumovirus] AAS22082.1 nucleoprotein [Humanmetapneumovirus] AHV79864.1 nucleoprotein [Human metapneumovirus]AHV79828.1 nucleoprotein [Human metapneumovirus] AGJ74084.1 nucleocapsidprotein [Human metapneumovirus] AAN52888.1 N [Human metapneumovirus][Human metapneumovirus] AIL23590.1 nucleoprotein [Human metapneumovirus]AAK62966.1 nucleoprotein [Human metapneumovirus] AHV79972.1nucleoprotein [Human metapneumovirus] AHV79470.1 nucleoprotein [Humanmetapneumovirus] AHV79452.1 nucleoprotein [Human metapneumovirus]AGJ74243.1 nucleoprotein [Human metapneumovirus] AHV79533.1nucleoprotein [Human metapneumovirus] AGJ74181.1 nucleoprotein [Humanmetapneumovirus] AHV79497.1 nucleoprotein [Human metapneumovirus]AHV79702.1 nucleoprotein [Human metapneumovirus] AHV79648.1nucleoprotein [Human metapneumovirus] AHV79435.1 putative nucleoprotein[Human metapneumovirus] AGJ74260.1 nucleocapsid protein [Humanmetapneumovirus] AAN52887.1 nucleoprotein [Human metapneumovirus]AGU68386.1 nucleocapsid protein [Human metapneumovirus] AAN52899.1nucleoprotein [Human metapneumovirus] AAR17673.1 nucleocapsid protein[Human metapneumovirus] AAN52898.1 nucleoprotein [Human metapneumovirus]AEA02277.1 nucleoprotein [Human metapneumovirus] AHV79612.1nucleoprotein [Human metapneumovirus] AGU68416.1 nucleoprotein [Humanmetapneumovirus] AGU68408.1 nucleoprotein [Human metapneumovirus]AGU68370.1 nucleoprotein [Human metapneumovirus] AAQ67683.1nucleoprotein [Human metapneumovirus] AGJ74137.1 nucleoprotein [Humanmetapneumovirus] AGU68344.1 nucleocapsid protein [Human metapneumovirus]ABK96997.1 nucleoprotein [Human metapneumovirus] AGU68413.1 nucleocapsidprotein [Human metapneumovirus] AAN52891.1 nucleoprotein [Humanmetapneumovirus] AGU68360.1 nucleoprotein [Human metapneumovirus]AGU68353.1 nucleocapsid protein [Human metapneumovirus] ABK96996.1nucleoprotein [Human metapneumovirus] AAR17666.1 N [Humanmetapneumovirus] [Human metapneumovirus] AEK26903.1 nucleoprotein [Humanmetapneumovirus] AGT75039.1 nucleoprotein [Human metapneumovirus]AGU68410.1 nucleoprotein [Human metapneumovirus] AAS22074.1nucleoprotein [Human metapneumovirus] AHV79560.1 nucleoprotein [Humanmetapneumovirus] AGT74978.1 nucleoprotein [Human metapneumovirus]AGJ74128.1 nucleoprotein [Human metapneumovirus] AAR17663.1nucleoprotein [Human metapneumovirus] AAR17662.1 nucleoprotein [Humanmetapneumovirus] AAR17664.1 nucleoprotein [Human metapneumovirus]AAR17657.1 nucleoprotein [Human metapneumovirus] AAR17659.1nucleoprotein [Human metapneumovirus] AAR17661.1 nucleoprotein [Humanmetapneumovirus] AGU68352.1 nucleoprotein [Human metapneumovirus]AGU68373.1 nucleoprotein [Human metapneumovirus] AGU68376.1nucleoprotein [Human metapneumovirus] AGU68342.1 nucleoprotein [Humanmetapneumovirus] AGU68365.1 nucleoprotein [Human metapneumovirus]AGU68363.1 nucleoprotein [Human metapneumovirus] AGU68398.1nucleoprotein [Human metapneumovirus] AGU68348.1 nucleoprotein [Humanmetapneumovirus] AGU68354.1 nucleoprotein [Human metapneumovirus]AGU68391.1 nucleoprotein [Human metapneumovirus] AGU68389.1nucleoprotein [Human metapneumovirus] AGU68399.1 nucleoprotein [Humanmetapneumovirus] AGU68337.1 nucleoprotein [Human metapneumovirus]AAR17660.1 nucleoprotein [Human metapneumovirus] AAR17667.1nucleoprotein [Human metapneumovirus] AGU68402.1 nucleoprotein [Avianmetapneumovirus type C] CDN30025.1 nucleoprotein [Avian metapneumovirus]AGZ87947.1 Nucleoprotein [Avian metapneumovirus type C] CAL25113.1nucleocapsid protein [Avian metapneumovirus] ABO42286.1 nucleocapsidprotein [Avian metapneumovirus] AAK38430.1 nucleocapsid protein [Avianmetapneumovirus] AAK54155.1 nucleocapsid protein [Avian metapneumovirus]AAK38426.1 nucleocapsid protein [Avian metapneumovirus] AAK38425.1nucleocapsid protein [Avian metapneumovirus] AAK38424.1 nucleocapsidprotein [Avian metapneumovirus] AAF05909.1 nucleocapsid protein [Avianmetapneumovirus] AAK38435.1 nucleocapsid protein [Avian metapneumovirus]AAK38428.1 nucleoprotein [Human metapneumovirus] AAR17669.1 nucleocapsidprotein [Avian metapneumovirus] AAK38429.1 nucleocapsid protein [Avianmetapneumovirus] AAK38427.1 nucleocapsid protein [Avian metapneumovirus]AAK38423.1 nucleocapsid protein [Avian metapneumovirus] AAK38434.1nucleoprotein [Human metapneumovirus] AGU68338.1 nucleoprotein [Avianmetapneumovirus] YP_443837.1 nucleoprotein [Human metapneumovirus]AGU68384.1 nucleocapsid protein [Avian metapneumovirus] AAK38431.1nucleoprotein [Human metapneumovirus] AGU68405.1 nucleoprotein [Humanmetapneumovirus] AGU68382.1 nucleoprotein [Human metapneumovirus]AGU68395.1 nucleocapsid [Human metapneumovirus] AAL35389.3 nucleoprotein[Human metapneumovirus] AEZ68064.1

TABLE 5  SEQ ID Description Sequence NO:PIV3 Nucleic Acid Sequences >gb|KJ672601.1|: ATGCCAATTTCAATACTGTTAATTATTACAACCATGATC 9 4990-6609 HumanATGGCATCACACTGCCAAATAGACATCACAAAACTACA parainfluenza virusGCATGTAGGTGTATTGGTCAACAGTCCCAAAGGGATGA 3 strainAGATATCACAAAACTTCGAAACAAGATATCTAATCCTGA HPIV3/Homo sapiens/GTCTCATACCAAAAATAGAAGATTCTAACTCTTGTGGTG PER/FLA4815/2008 ACCAACAGATCAAGCAATACAAGAGGTTATTGGATAGA [fusion glycoproteinCTGATCATTCCTTTATATGATGGACTAAGATTACAGAAG F0]GATGTGATAGTGACTAATCAAGAATCCAATGAAAACACTGATCCCAGAACAGAACGATTCTTTGGAGGGGTAATTGGAACTATTGCTCTAGGAGTAGCAACCTCAGCACAAATTACAGCAGCAGTTGCTCTGGTTGAAGCCAAGCAGGCAAGATCAGACATTGAAAAACTCAAGGAAGCAATCAGGGACACAAATAAAGCAGTGCAGTCAGTTCAGAGCTCTGTAGGAAATTTGATAGTAGCAATTAAATCAGTCCAGGATTATGTCAACAAAGAAATCGTGCCATCGATTGCGAGACTAGGTTGTGAAGCAGCAGGACTTCAGTTAGGGATTGCATTAACACAGCATTACTCAGAATTAACAAATATATTTGGTGATAACATAGGATCGTTACAAGAAAAAGGAATAAAATTACAAGGTATAGCATCATTATACCGTACAAATATCACAGAAATATTCACAACATCAACAGTTGACAAATATGATATTTATGATCTATTATTTACAGAATCAATAAAGGTGAGAGTTATAGATGTTGATTTGAATGATTACTCAATAACCCTCCAAGTCAGACTCCCTTTATTGACCAGACTGCTGAACACTCAAATCTACAAAGTAGATTCCATATCATACAATATCCAAAATAGAGAATGGTATATCCCTCTTCCCAGCCATATCATGACGAAAGGGGCATTTCTAGGTGGAGCAGATGTCAAAGAATGCATAGAAGCATTCAGCAGTTATATATGCCCTTCTGATCCAGGATTTGTACTAAACCATGAAATGGAGAGCTGTCTATCAGGAAACATATCCCAATGTCCAAGAACCACAGTCACATCAGACATAGTTCCTAGGTATGCATTTGTCAATGGAGGAGTGGTTGCGAATTGTATAACAACTACATGTACATGCAATGGTATCGGTAATAGAATCAACCAACCACCTGATCAAGGAGTCAAAATTATAACACATAAAGAATGTAATACAATAGGTATCAACGGAATGCTATTCAACACAAACAAAGAAGGAACTCTTGCATTCTACACACCAGACGACATAACATTAAACAATTCTGTTGCACTTGATCCGATTGACATATCAATCGAGCTCAACAAGGCCAAATCAGATCTTGAGGAATCAAAAGAATGGATAAGAAGGTCAAATCAAAAGCTAGATTCTATTGGAAGTTGGCATCAATCTAGCACTACAATCATAGTTATTTTGATAATGATGATTATATTGTTTATAATTAATATAACAATAATTACAATTGCAATTAAGTATTACAGAATTCAAAAGAGAAATCGAGTGGAT CAAAATGATAAGCCGTATGTATTAACAAACAAGgi|612507167|gb| ATGGAATACTGGAAGCACACCAACCACGGAAAGGATGC 10 AHX22430.1|TGGTAATGAGCTGGAGACATCCACAGCCACTCATGGCA hemagglutinin-ACAAGCTCACCAACAAGATAACATATATATTGTGGACG neuraminidaseATAACCCTGGTGTTATTATCAATAGTCTTCATCATAGTG [Human parainfluenzaCTAACTAATTCCATCAAAAGTGAAAAGGCCCGCGAATC virus 3]ATTGCTACAAGACATAAATAATGAGTTTATGGAAGTTACAGAAAAGATCCAAGTGGCATCGGATAATACTAATGATCTAATACAGTCAGGAGTGAATACAAGGCTTCTTACAATTCAGAGTCATGTCCAGAATTATATACCAATATCATTGACACAACAAATATCGGATCTTAGGAAATTCATTAGTGAAATTACAATTAGAAATGATAATCAAGAAGTGCCACCACAAAGAATAACACATGATGTGGGTATAAAACCTTTAAATCCAGATGATTTCTGGAGATGCACGTCTGGTCTTCCATCTTTGATGAAAACTCCAAAAATAAGATTAATGCCGGGACCAGGATTATTAGCTATGCCAACGACTGTTGATGGCTGTGTCAGAACCCCGTCCTTAGTGATAAATGATCTGATTTATGCTTACACCTCAAATCTAATTACTCGAGGTTGCCAGGATATAGGGAAATCATATCAAGTATTACAGATAGGGATAATAACTGTAAACTCAGACTTGGTACCTGACTTAAATCCTAGGATCTCTCATACCTTCAACATAAATGACAATAGAAAGTCATGTTCTCTAGCACTCCTAAATACAGATGTATATCAACTGTGTTCAACCCCAAAAGTTGATGAAAGATCAGATTATGCATCATCAGGCATAGAAGATATTGTACTTGATATTGTCAATTATGATGGCTCAATCTCGACAACAAGATTTAAGAATAATAATATAAGTTTTGATCAACCATATGCGGCATTATACCCATCTGTTGGACCAGGGATATACTACAAAGGCAAAATAATATTTCTCGGGTATGGAGGTCTTGAACATCCAATAAATGAGAATGCAATCTGCAACACAACTGGGTGTCCTGGGAAAACACAGAGAGACTGTAATCAAGCATCTCATAGTCCATGGTTTTCAGATAGAAGGATGGTCAACTCTATAATTGTTGTTGACAAGGGCTTGAACTCAGTTCCAAAATTGAAGGTATGGACGATATCTATGAGACAAAATTACTGGGGGTCAGAAGGAAGATTACTTCTACTAGGTAACAAGATCTACATATACACAAGATCTACAAGTTGGCACAGCAAGTTACAATTAGGAATAATTGACATTACTGACTACAGTGATATAAGGATAAAATGGACATGGCATAATGTGCTATCAAGACCAGGAAACAATGAATGTCCATGGGGACATTCATGTCCGGATGGATGTATAACGGGAGTATATACCGATGCATATCCACTCAATCCCACAGGAAGCATTGTATCATCTGTCATATTGGACTCACAAAAATCGAGAGTCAACCCAGTCATAACTTACTCAACAGCAACCGAAAGGGTAAACGAGCTGGCTATCCGAAACAAAACACTCTCAGCTGGGTACACAACAACAAGCTGCATTACACACTATAACAAAGGGTATTGTTTTCATATAGTAGAAATAAATCATAAAAGCTTAAACACATTTCAACCCATGTTGTTCAAAACAGA GATTCCAAAAAGCTGCAGTHPIV3_HN_Codon ATGGAATACTGGAAGCACACCAACCACGGCAAGGACGC 11 OptimizedCGGCAACGAGCTGGAAACCAGCACAGCCACACACGGCAACAAGCTGACCAACAAGATCACCTACATCCTGTGGACCATCACCCTGGTGCTGCTGAGCATCGTGTTCATCATCGTGCTGACCAATAGCATCAAGAGCGAGAAGGCCAGAGAGAGCCTGCTGCAGGACATCAACAACGAGTTCATGGAAGTGACCGAGAAGATCCAGGTGGCCAGCGACAACACCAACGACCTGATCCAGAGCGGCGTGAACACCCGGCTGCTGACCATCCAGAGCCACGTGCAGAACTACATCCCCATCAGCCTGACCCAGCAGATCAGCGACCTGCGGAAGTTCATCAGCGAGATCACCATCCGGAACGACAACCAGGAAGTGCCCCCCCAGAGAATCACCCACGACGTGGGCATCAAGCCCCTGAACCCCGACGATTTCTGGCGGTGTACAAGCGGCCTGCCCAGCCTGATGAAGACCCCCAAGATCCGGCTGATGCCTGGCCCTGGACTGCTGGCCATGCCTACCACAGTGGATGGCTGTGTGCGGACCCCCAGCCTCGTGATCAACGATCTGATCTACGCCTACACCAGCAACCTGATCACCCGGGGCTGCCAGGATATCGGCAAGAGCTACCAGGTGCTGCAGATCGGCATCATCACCGTGAACTCCGACCTGGTGCCCGACCTGAACCCTCGGATCAGCCACACCTTCAACATCAACGACAACAGAAAGAGCTGCAGCCTGGCTCTGCTGAACACCGACGTGTACCAGCTGTGCAGCACCCCCAAGGTGGACGAGAGAAGCGACTACGCCAGCAGCGGCATCGAGGATATCGTGCTGGACATCGTGAACTACGACGGCAGCATCAGCACCACCCGGTTCAAGAACAACAACATCAGCTTCGACCAGCCCTACGCCGCCCTGTACCCTTCTGTGGGCCCTGGCATCTACTACAAGGGCAAGATCATCTTCCTGGGCTACGGCGGCCTGGAACACCCCATCAACGAGAACGCCATCTGCAACACCACCGGCTGCCCTGGCAAGACCCAGAGAGACTGCAATCAGGCCAGCCACAGCCCCTGGTTCAGCGACCGCAGAATGGTCAACTCTATCATCGTGGTGGACAAGGGCCTGAACAGCGTGCCCAAGCTGAAAGTGTGGACAATCAGCATGCGCCAGAACTACTGGGGCAGCGAGGGCAGACTTCTGCTGCTGGGAAACAAGATCTACATCTACACCCGGTCCACCAGCTGGCACAGCAAACTGCAGCTGGGAATCATCGACATCACCGACTACAGCGACATCCGGATCAAGTGGACCTGGCACAACGTGCTGAGCAGACCCGGCAACAATGAGTGCCCTTGGGGCCACAGCTGCCCCGATGGATGTATCACCGGCGTGTACACCGACGCCTACCCCCTGAATCCTACCGGCTCCATCGTGTCCAGCGTGATCCTGGACAGCCAGAAAAGCAGAGTGAACCCCGTGATCACATACAGCACCGCCACCGAGAGAGTGAACGAACTGGCCATCAGAAACAAGACCCTGAGCGCCGGCTACACCACCACAAGCTGCATCACACACTACAACAAGGGCTACTGCTTCCACATCGTGGAAATCAACCACAAGTCCCTGAACACCTTCCAGCCCATGCTGTTCA AGACCGAGATCCCCAAGAGCTGCTCCHPIV3_F_Codon ATGCCCATCAGCATCCTGCTGATCATCACCACAATGATC 12 OptimizedATGGCCAGCCACTGCCAGATCGACATCACCAAGCTGCAGCACGTGGGCGTGCTCGTGAACAGCCCCAAGGGCATGAAGATCAGCCAGAACTTCGAGACACGCTACCTGATCCTGAGCCTGATCCCCAAGATCGAGGACAGCAACAGCTGCGGCGACCAGCAGATCAAGCAGTACAAGCGGCTGCTGGACAGACTGATCATCCCCCTGTACGACGGCCTGCGGCTGCAGAAAGACGTGATCGTGACCAACCAGGAAAGCAACGAGAACACCGACCCCCGGACCGAGAGATTCTTCGGCGGCGTGATCGGCACAATCGCCCTGGGAGTGGCCACAAGCGCCCAGATTACAGCCGCTGTGGCCCTGGTGGAAGCCAAGCAGGCCAGAAGCGACATCGAGAAGCTGAAAGAGGCCATCCGGGACACCAACAAGGCCGTGCAGAGCGTGCAGTCCAGCGTGGGCAATCTGATCGTGGCCATCAAGTCCGTGCAGGACTACGTGAACAAAGAAATCGTGCCCTCTATCGCCCGGCTGGGCTGTGAAGCTGCCGGACTGCAGCTGGGCATTGCCCTGACACAGCACTACAGCGAGCTGACCAACATCTTCGGCGACAACATCGGCAGCCTGCAGGAAAAGGGCATTAAGCTGCAGGGAATCGCCAGCCTGTACCGCACCAACATCACCGAGATCTTCACCACCAGCACCGTGGATAAGTACGACATCTACGACCTGCTGTTCACCGAGAGCATCAAAGTGCGCGTGATCGACGTGGACCTGAACGACTACAGCATCACCCTGCAAGTGCGGCTGCCCCTGCTGACCAGACTGCTGAACACCCAGATCTACAAGGTGGACAGCATCTCCTACAACATCCAGAACCGCGAGTGGTACATCCCTCTGCCCAGCCACATTATGACCAAGGGCGCCTTTCTGGGCGGAGCCGACGTGAAAGAGTGCATCGAGGCCTTCAGCAGCTACATCTGCCCCAGCGACCCTGGCTTCGTGCTGAACCACGAGATGGAAAGCTGCCTGAGCGGCAACATCAGCCAGTGCCCCAGAACCACCGTGACCTCCGACATCGTGCCCAGATACGCCTTCGTGAATGGCGGCGTGGTGGCCAACTGCATCACCACCACCTGTACCTGCAACGGCATCGGCAACCGGATCAACCAGCCTCCCGATCAGGGCGTGAAGATTATCACCCACAAAGAGTGTAACACCATCGGCATCAACGGCATGCTGTTCAATACCAACAAAGAGGGCACCCTGGCCTTCTACACCCCCGACGATATCACCCTGAACAACTCCGTGGCTCTGGACCCCATCGACATCTCCATCGAGCTGAACAAGGCCAAGAGCGACCTGGAAGAGTCCAAAGAGTGGATCCGGCGGAGCAACCAGAAGCTGGACTCTATCGGCAGCTGGCACCAGAGCAGCACCACCATCATCGTGATCCTGATTATGATGATTATCCTGTTCATCATCAACATTACCATCATCACTATCGCCATTAAGTACTACCGGATCCAGAAACGGAACCGGGTGGACCAGAATGACAAGCCCTACGTGCTGACAAAC AAGPIV3 mRNA Sequences >gb|KJ672601.1|: AUGCCAAUUUCAAUACUGUUAAUUAUUACAACCAUGA 61 4990-6609 HumanUCAUGGCAUCACACUGCCAAAUAGACAUCACAAAACU parainfluenza virusACAGCAUGUAGGUGUAUUGGUCAACAGUCCCAAAGGG 3 strainAUGAAGAUAUCACAAAACUUCGAAACAAGAUAUCUAA HPIV3/Homo sapiens/UCCUGAGUCUCAUACCAAAAAUAGAAGAUUCUAACUC PER/FLA4815/UUGUGGUGACCAACAGAUCAAGCAAUACAAGAGGUUA 2008 [fusionUUGGAUAGACUGAUCAUUCCUUUAUAUGAUGGACUAA glycoprotein F0]GAUUACAGAAGGAUGUGAUAGUGACUAAUCAAGAAUCCAAUGAAAACACUGAUCCCAGAACAGAACGAUUCUUUGGAGGGGUAAUUGGAACUAUUGCUCUAGGAGUAGCAACCUCAGCACAAAUUACAGCAGCAGUUGCUCUGGUUGAAGCCAAGCAGGCAAGAUCAGACAUUGAAAAACUCAAGGAAGCAAUCAGGGACACAAAUAAAGCAGUGCAGUCAGUUCAGAGCUCUGUAGGAAAUUUGAUAGUAGCAAUUAAAUCAGUCCAGGAUUAUGUCAACAAAGAAAUCGUGCCAUCGAUUGCGAGACUAGGUUGUGAAGCAGCAGGACUUCAGUUAGGGAUUGCAUUAACACAGCAUUACUCAGAAUUAACAAAUAUAUUUGGUGAUAACAUAGGAUCGUUACAAGAAAAAGGAAUAAAAUUACAAGGUAUAGCAUCAUUAUACCGUACAAAUAUCACAGAAAUAUUCACAACAUCAACAGUUGACAAAUAUGAUAUUUAUGAUCUAUUAUUUACAGAAUCAAUAAAGGUGAGAGUUAUAGAUGUUGAUUUGAAUGAUUACUCAAUAACCCUCCAAGUCAGACUCCCUUUAUUGACCAGACUGCUGAACACUCAAAUCUACAAAGUAGAUUCCAUAUCAUACAAUAUCCAAAAUAGAGAAUGGUAUAUCCCUCUUCCCAGCCAUAUCAUGACGAAAGGGGCAUUUCUAGGUGGAGCAGAUGUCAAAGAAUGCAUAGAAGCAUUCAGCAGUUAUAUAUGCCCUUCUGAUCCAGGAUUUGUACUAAACCAUGAAAUGGAGAGCUGUCUAUCAGGAAACAUAUCCCAAUGUCCAAGAACCACAGUCACAUCAGACAUAGUUCCUAGGUAUGCAUUUGUCAAUGGAGGAGUGGUUGCGAAUUGUAUAACAACUACAUGUACAUGCAAUGGUAUCGGUAAUAGAAUCAACCAACCACCUGAUCAAGGAGUCAAAAUUAUAACACAUAAAGAAUGUAAUACAAUAGGUAUCAACGGAAUGCUAUUCAACACAAACAAAGAAGGAACUCUUGCAUUCUACACACCAGACGACAUAACAUUAAACAAUUCUGUUGCACUUGAUCCGAUUGACAUAUCAAUCGAGCUCAACAAGGCCAAAUCAGAUCUUGAGGAAUCAAAAGAAUGGAUAAGAAGGUCAAAUCAAAAGCUAGAUUCUAUUGGAAGUUGGCAUCAAUCUAGCACUACAAUCAUAGUUAUUUUGAUAAUGAUGAUUAUAUUGUUUAUAAUUAAUAUAACAAUAAUUACAAUUGCAAUUAAGUAUUACAGAAUUCAAAAGAGAAAUCGAGUGGAUCAAAAUG AUAAGCCGUAUGUAUUAACAAACAAGgi|612507167|gb| AUGGAAUACUGGAAGCACACCAACCACGGAAAGGAUG 62 AHX22430.1|CUGGUAAUGAGCUGGAGACAUCCACAGCCACUCAUGG hemagglutinin-CAACAAGCUCACCAACAAGAUAACAUAUAUAUUGUGG neuraminidaseACGAUAACCCUGGUGUUAUUAUCAAUAGUCUUCAUCA [HumanUAGUGCUAACUAAUUCCAUCAAAAGUGAAAAGGCCCG parainfluenza virusCGAAUCAUUGCUACAAGACAUAAAUAAUGAGUUUAUG 3]GAAGUUACAGAAAAGAUCCAAGUGGCAUCGGAUAAUACUAAUGAUCUAAUACAGUCAGGAGUGAAUACAAGGCUUCUUACAAUUCAGAGUCAUGUCCAGAAUUAUAUACCAAUAUCAUUGACACAACAAAUAUCGGAUCUUAGGAAAUUCAUUAGUGAAAUUACAAUUAGAAAUGAUAAUCAAGAAGUGCCACCACAAAGAAUAACACAUGAUGUGGGUAUAAAACCUUUAAAUCCAGAUGAUUUCUGGAGAUGCACGUCUGGUCUUCCAUCUUUGAUGAAAACUCCAAAAAUAAGAUUAAUGCCGGGACCAGGAUUAUUAGCUAUGCCAACGACUGUUGAUGGCUGUGUCAGAACCCCGUCCUUAGUGAUAAAUGAUCUGAUUUAUGCUUACACCUCAAAUCUAAUUACUCGAGGUUGCCAGGAUAUAGGGAAAUCAUAUCAAGUAUUACAGAUAGGGAUAAUAACUGUAAACUCAGACUUGGUACCUGACUUAAAUCCUAGGAUCUCUCAUACCUUCAACAUAAAUGACAAUAGAAAGUCAUGUUCUCUAGCACUCCUAAAUACAGAUGUAUAUCAACUGUGUUCAACCCCAAAAGUUGAUGAAAGAUCAGAUUAUGCAUCAUCAGGCAUAGAAGAUAUUGUACUUGAUAUUGUCAAUUAUGAUGGCUCAAUCUCGACAACAAGAUUUAAGAAUAAUAAUAUAAGUUUUGAUCAACCAUAUGCGGCAUUAUACCCAUCUGUUGGACCAGGGAUAUACUACAAAGGCAAAAUAAUAUUUCUCGGGUAUGGAGGUCUUGAACAUCCAAUAAAUGAGAAUGCAAUCUGCAACACAACUGGGUGUCCUGGGAAAACACAGAGAGACUGUAAUCAAGCAUCUCAUAGUCCAUGGUUUUCAGAUAGAAGGAUGGUCAACUCUAUAAUUGUUGUUGACAAGGGCUUGAACUCAGUUCCAAAAUUGAAGGUAUGGACGAUAUCUAUGAGACAAAAUUACUGGGGGUCAGAAGGAAGAUUACUUCUACUAGGUAACAAGAUCUACAUAUACACAAGAUCUACAAGUUGGCACAGCAAGUUACAAUUAGGAAUAAUUGACAUUACUGACUACAGUGAUAUAAGGAUAAAAUGGACAUGGCAUAAUGUGCUAUCAAGACCAGGAAACAAUGAAUGUCCAUGGGGACAUUCAUGUCCGGAUGGAUGUAUAACGGGAGUAUAUACCGAUGCAUAUCCACUCAAUCCCACAGGAAGCAUUGUAUCAUCUGUCAUAUUGGACUCACAAAAAUCGAGAGUCAACCCAGUCAUAACUUACUCAACAGCAACCGAAAGGGUAAACGAGCUGGCUAUCCGAAACAAAACACUCUCAGCUGGGUACACAACAACAAGCUGCAUUACACACUAUAACAAAGGGUAUUGUUUUCAUAUAGUAGAAAUAAAUCAUAAAAGCUUAAACACAUUUCAACCCAUGUUGUUCAAAACAGAGAUUC CAAAAAGCUGCAGU HPIV3_HN_CodonAUGGAAUACUGGAAGCACACCAACCACGGCAAGGACG 63 OptimizedCCGGCAACGAGCUGGAAACCAGCACAGCCACACACGGCAACAAGCUGACCAACAAGAUCACCUACAUCCUGUGGACCAUCACCCUGGUGCUGCUGAGCAUCGUGUUCAUCAUCGUGCUGACCAAUAGCAUCAAGAGCGAGAAGGCCAGAGAGAGCCUGCUGCAGGACAUCAACAACGAGUUCAUGGAAGUGACCGAGAAGAUCCAGGUGGCCAGCGACAACACCAACGACCUGAUCCAGAGCGGCGUGAACACCCGGCUGCUGACCAUCCAGAGCCACGUGCAGAACUACAUCCCCAUCAGCCUGACCCAGCAGAUCAGCGACCUGCGGAAGUUCAUCAGCGAGAUCACCAUCCGGAACGACAACCAGGAAGUGCCCCCCCAGAGAAUCACCCACGACGUGGGCAUCAAGCCCCUGAACCCCGACGAUUUCUGGCGGUGUACAAGCGGCCUGCCCAGCCUGAUGAAGACCCCCAAGAUCCGGCUGAUGCCUGGCCCUGGACUGCUGGCCAUGCCUACCACAGUGGAUGGCUGUGUGCGGACCCCCAGCCUCGUGAUCAACGAUCUGAUCUACGCCUACACCAGCAACCUGAUCACCCGGGGCUGCCAGGAUAUCGGCAAGAGCUACCAGGUGCUGCAGAUCGGCAUCAUCACCGUGAACUCCGACCUGGUGCCCGACCUGAACCCUCGGAUCAGCCACACCUUCAACAUCAACGACAACAGAAAGAGCUGCAGCCUGGCUCUGCUGAACACCGACGUGUACCAGCUGUGCAGCACCCCCAAGGUGGACGAGAGAAGCGACUACGCCAGCAGCGGCAUCGAGGAUAUCGUGCUGGACAUCGUGAACUACGACGGCAGCAUCAGCACCACCCGGUUCAAGAACAACAACAUCAGCUUCGACCAGCCCUACGCCGCCCUGUACCCUUCUGUGGGCCCUGGCAUCUACUACAAGGGCAAGAUCAUCUUCCUGGGCUACGGCGGCCUGGAACACCCCAUCAACGAGAACGCCAUCUGCAACACCACCGGCUGCCCUGGCAAGACCCAGAGAGACUGCAAUCAGGCCAGCCACAGCCCCUGGUUCAGCGACCGCAGAAUGGUCAACUCUAUCAUCGUGGUGGACAAGGGCCUGAACAGCGUGCCCAAGCUGAAAGUGUGGACAAUCAGCAUGCGCCAGAACUACUGGGGCAGCGAGGGCAGACUUCUGCUGCUGGGAAACAAGAUCUACAUCUACACCCGGUCCACCAGCUGGCACAGCAAACUGCAGCUGGGAAUCAUCGACAUCACCGACUACAGCGACAUCCGGAUCAAGUGGACCUGGCACAACGUGCUGAGCAGACCCGGCAACAAUGAGUGCCCUUGGGGCCACAGCUGCCCCGAUGGAUGUAUCACCGGCGUGUACACCGACGCCUACCCCCUGAAUCCUACCGGCUCCAUCGUGUCCAGCGUGAUCCUGGACAGCCAGAAAAGCAGAGUGAACCCCGUGAUCACAUACAGCACCGCCACCGAGAGAGUGAACGAACUGGCCAUCAGAAACAAGACCCUGAGCGCCGGCUACACCACCACAAGCUGCAUCACACACUACAACAAGGGCUACUGCUUCCACAUCGUGGAAAUCAACCACAAGUCCCUGAACACCUUCCAGCCCAUGCUGUU CAAGACCGAGAUCCCCAAGAGCUGCUCCHPIV3_F_Codon AUGCCCAUCAGCAUCCUGCUGAUCAUCACCACAAUGAU 64 Optimized mRNACAUGGCCAGCCACUGCCAGAUCGACAUCACCAAGCUGC sequenceAGCACGUGGGCGUGCUCGUGAACAGCCCCAAGGGCAUGAAGAUCAGCCAGAACUUCGAGACACGCUACCUGAUCCUGAGCCUGAUCCCCAAGAUCGAGGACAGCAACAGCUGCGGCGACCAGCAGAUCAAGCAGUACAAGCGGCUGCUGGACAGACUGAUCAUCCCCCUGUACGACGGCCUGCGGCUGCAGAAAGACGUGAUCGUGACCAACCAGGAAAGCAACGAGAACACCGACCCCCGGACCGAGAGAUUCUUCGGCGGCGUGAUCGGCACAAUCGCCCUGGGAGUGGCCACAAGCGCCCAGAUUACAGCCGCUGUGGCCCUGGUGGAAGCCAAGCAGGCCAGAAGCGACAUCGAGAAGCUGAAAGAGGCCAUCCGGGACACCAACAAGGCCGUGCAGAGCGUGCAGUCCAGCGUGGGCAAUCUGAUCGUGGCCAUCAAGUCCGUGCAGGACUACGUGAACAAAGAAAUCGUGCCCUCUAUCGCCCGGCUGGGCUGUGAAGCUGCCGGACUGCAGCUGGGCAUUGCCCUGACACAGCACUACAGCGAGCUGACCAACAUCUUCGGCGACAACAUCGGCAGCCUGCAGGAAAAGGGCAUUAAGCUGCAGGGAAUCGCCAGCCUGUACCGCACCAACAUCACCGAGAUCUUCACCACCAGCACCGUGGAUAAGUACGACAUCUACGACCUGCUGUUCACCGAGAGCAUCAAAGUGCGCGUGAUCGACGUGGACCUGAACGACUACAGCAUCACCCUGCAAGUGCGGCUGCCCCUGCUGACCAGACUGCUGAACACCCAGAUCUACAAGGUGGACAGCAUCUCCUACAACAUCCAGAACCGCGAGUGGUACAUCCCUCUGCCCAGCCACAUUAUGACCAAGGGCGCCUUUCUGGGCGGAGCCGACGUGAAAGAGUGCAUCGAGGCCUUCAGCAGCUACAUCUGCCCCAGCGACCCUGGCUUCGUGCUGAACCACGAGAUGGAAAGCUGCCUGAGCGGCAACAUCAGCCAGUGCCCCAGAACCACCGUGACCUCCGACAUCGUGCCCAGAUACGCCUUCGUGAAUGGCGGCGUGGUGGCCAACUGCAUCACCACCACCUGUACCUGCAACGGCAUCGGCAACCGGAUCAACCAGCCUCCCGAUCAGGGCGUGAAGAUUAUCACCCACAAAGAGUGUAACACCAUCGGCAUCAACGGCAUGCUGUUCAAUACCAACAAAGAGGGCACCCUGGCCUUCUACACCCCCGACGAUAUCACCCUGAACAACUCCGUGGCUCUGGACCCCAUCGACAUCUCCAUCGAGCUGAACAAGGCCAAGAGCGACCUGGAAGAGUCCAAAGAGUGGAUCCGGCGGAGCAACCAGAAGCUGGACUCUAUCGGCAGCUGGCACCAGAGCAGCACCACCAUCAUCGUGAUCCUGAUUAUGAUGAUUAUCCUGUUCAUCAUCAACAUUACCAUCAUCACUAUCGCCAUUAAGUACUACCGGAUCCAGAAACGGAACCGGGUGGACCAGAAUGACAAGCCCUACGUGCUG ACAAACAAG

TABLE 6 PIV3 Amino Acid Sequences SEQ ID Description SequenceNO: >gi|612507166|gb| MPISILLIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQ 13AHX22429.1| NFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDG fusionLRLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSA glycoproteinQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSVG F0 [HumanNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYS parainfluenza ELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKY virus 3]DIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGSWHQSSTTIIVILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK gi|612507167|gb|MEYWKHTNHGKDAGNELETSTATHGNKLTNKITYILWTIT 14 AHX22430.1|LVLLSIVFIIVLTNSIKSEKARESLLQDINNEFMEVTEKIQVA hemagglutinin-SDNTNDLIQSGVNTRLLTIQSHVQNYIPISLTQQISDLRKFIS neuraminidaseEITIRNDNQEVPPQRITHDVGIKPLNPDDFWRCTSGLPSLMK [HumanTPKIRLMPGPGLLAMPTTVDGCVRTPSLVINDLIYAYTSNLI parainfluenzaTRGCQDIGKSYQVLQIGIITVNSDLVPDLNPRISHTFNINDN virus 3]RKSCSLALLNTDVYQLCSTPKVDERSDYASSGIEDIVLDIVNYDGSISTTRFKNNNISFDQPYAALYPSVGPGIYYKGKIIFLGYGGLEHPINENAICNTTGCPGKTQRDCNQASHSPWFSDRRMVNSIIVVDKGLNSVPKLKVWTISMRQNYWGSEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITDYSDIRIKWTWHNVLSRPGNNECPWGHSCPDGCITGVYTDAYPLNPTGSIVSSVILDSQKSRVNPVITYSTATERVNELAIRNKTLSAGYTTTSCITHYNKGYCFHIVEINHKSLNTFQPMLFKTEIPKSCS

TABLE 7 PIV3 NCBI Accession Numbers (Nucleic Acid and Amino AcidSequences) Description GenBank Accession Fusion glycoprotein F0 [Humanparainfluenza virus 3] KJ672601.1|: HPIV3/Homo sapiens/PER/FLA4815/20084990-6609 AHX22429 (Fusion protein) hemagglutinin-neuraminidase [Humanparainfluenza virus 3] KJ672601.1|: HPIV3/Homo sapiens/PER/FLA4815/20086724-8442 AHX22430 (HN protein) Recombinant PIV3/PIV1 virus fusionglycoprotein (F) AF016281 and hemagglutinin (HN) genes, complete cds;and RNA AAC23947 dependent RNA polymerase (L) gene, partial cds.(hemagglutinin) Recombinant PIV3/PIV1 virus fusion glycoprotein (F)AF016281 and hemagglutinin (HN) genes, complete cds; and RNA AAC23947dependent RNA polymerase (L) gene, partial cds. (fusion protein)hemagglutinin-neuraminidase [Human parainfluenza virus 3] BAO32044.1hemagglutinin-neuraminidase [Human parainfluenza virus 3] BAO32051.1 Cprotein [Human parainfluenza virus 3] NP_599251.1 C protein [Humanparainfluenza virus 3] ABZ85670.1 C protein [Human parainfluenza virus3] AGT75164.1 C protein [Human parainfluenza virus 3] AAB48686.1 Cprotein [Human parainfluenza virus 3] AHX22115.1 C protein [Humanparainfluenza virus 3] AGW51066.1 C protein [Human parainfluenza virus3] AGW51162.1 C protein [Human parainfluenza virus 3] AGT75252.1 Cprotein [Human parainfluenza virus 3] AGT75188.1 C protein [Humanparainfluenza virus 3] AGW51218.1 C protein [Human parainfluenza virus3] AGW51074.1 C protein [Human parainfluenza virus 3] AGT75323.1 Cprotein [Human parainfluenza virus 3] AGT75307.1 C protein [Humanparainfluenza virus 3] AHX22131.1 C protein [Human parainfluenza virus3] AGW51243.1 C protein [Human parainfluenza virus 3] AGT75180.1 Cprotein [Human parainfluenza virus 3] AGT75212.1 C protein [Humanparainfluenza virus 3] AGW51186.1 C protein [Human parainfluenza virus3] AHX22075.1 C protein [Human parainfluenza virus 3] AHX22163.1 Cprotein [Human parainfluenza virus 3] AGT75196.1 C protein [Humanparainfluenza virus 3] AHX22491.1 C protein [Human parainfluenza virus3] AHX22139.1 C protein [Human parainfluenza virus 3] AGW51138.1 Cprotein [Human parainfluenza virus 3] AGW51114.1 C protein [Humanparainfluenza virus 3] AGT75220.1 C protein [Human parainfluenza virus3] AHX22251.1 RecName: Full = Protein C; AltName: Full = VP18 proteinP06165.1 C protein [Human parainfluenza virus 3] AHX22187.1 C protein[Human parainfluenza virus 3] AGT75228.1 C protein [Human parainfluenzavirus 3] AHX22179.1 C protein [Human parainfluenza virus 3] AHX22427.1 Cprotein [Human parainfluenza virus 3] AGW51210.1 nonstructural protein C[Human parainfluenza virus 3] BAA00922.1 C protein [Human parainfluenzavirus 3] AHX22315.1 C protein [Human parainfluenza virus 3] AGW51259.1 Cprotein [Human parainfluenza virus 3] AHX22435.1 C protein [Humanparainfluenza virus 3] AHX22123.1 C protein [Human parainfluenza virus3] AHX22299.1 C protein [Human parainfluenza virus 3] AGW51267.1 unnamedprotein product [Human parainfluenza virus 3] CAA28430.1 C protein[Human parainfluenza virus 3] AGW51178.1 C protein [Human parainfluenzavirus 3] AHX22411.1 RecName: Full = Protein C P06164.1 phosphoprotein[Human parainfluenza virus 3] NP_067149.1 phosphoprotein [Humanparainfluenza virus 3] AAB48685.1 phosphoprotein [Human parainfluenzavirus 3] AHX22498.1 phosphoprotein [Human parainfluenza virus 3]AHX22490.1 phosphoprotein [Human parainfluenza virus 3] AGT75259.1phosphoprotein [Human parainfluenza virus 3] AGW51137.1 phosphoprotein[Human parainfluenza virus 3] AGW51145.1 phosphoprotein [Humanparainfluenza virus 3] AGT75298.1 phosphoprotein [Human parainfluenzavirus 3] AGW51113.1 phosphoprotein [Human parainfluenza virus 3]AGT75203.1 phosphoprotein [Human parainfluenza virus 3] AGT75163.1phosphoprotein [Human parainfluenza virus 3] AHX22506.1 phosphoprotein[Human parainfluenza virus 3] AGW51129.1 phosphoprotein [Humanparainfluenza virus 3] AHX22194.1 phosphoprotein [Human parainfluenzavirus 3] AGT75211.1 phosphoprotein [Human parainfluenza virus 3]AHX22258.1 phosphoprotein [Human parainfluenza virus 3] AGW51121.1phosphoprotein [Human parainfluenza virus 3] AGT75282.1 phosphoprotein[Human parainfluenza virus 3] AHX22146.1 phosphoprotein [Humanparainfluenza virus 3] AHX22138.1 phosphoprotein [Human parainfluenzavirus 3] AHX22322.1 phosphoprotein [Human parainfluenza virus 3]AHX22370.1 phosphoprotein [Human parainfluenza virus 3] AHX22098.1phosphoprotein [Human parainfluenza virus 3] AHX22130.1 phosphoprotein[Human parainfluenza virus 3] AHX22418.1 phosphoprotein [Humanparainfluenza virus 3] AHX22114.1 phosphoprotein [Human parainfluenzavirus 3] AHX22410.1 phosphoprotein [Human parainfluenza virus 3]AGT75306.1 phosphoprotein [Human parainfluenza virus 3] AHX22170.1phosphoprotein [Human parainfluenza virus 3] AHX22266.1 phosphoprotein[Human parainfluenza virus 3] AHX22090.1 phosphoprotein [Humanparainfluenza virus 3] AGT75195.1 phosphoprotein [Human parainfluenzavirus 3] AHX22226.1 phosphoprotein [Human parainfluenza virus 3]AHX22178.1 phosphoprotein [Human parainfluenza virus 3] AHX22122.1phosphoprotein [Human parainfluenza virus 3] AHX22186.1 phosphoprotein[Human parainfluenza virus 3] AHX22066.1 phosphoprotein [Humanparainfluenza virus 3] AHX22522.1 phosphoprotein [Human parainfluenzavirus 3] AGW51225.1 phosphoprotein [Human parainfluenza virus 3]BAN29032.1 phosphoprotein [Human parainfluenza virus 3] ABZ85669.1phosphoprotein [Human parainfluenza virus 3] AHX22426.1 phosphoprotein[Human parainfluenza virus 3] AHX22058.1 phosphoprotein [Simian Agent10] ADR00400.1 phosphoprotein [Human parainfluenza virus 3] AHX22250.1phosphoprotein [Human parainfluenza virus 3] AHX22434.1 phosphoprotein[Human parainfluenza virus 3] AHX22298.1 phosphoprotein [Humanparainfluenza virus 3] AHX22442.1 phosphoprotein [Human parainfluenzavirus 3] AHX22074.1 phosphoprotein [Human parainfluenza virus 3]AGW51153.1 phosphoprotein [Human parainfluenza virus 3] AGW51241.1phosphoprotein [Human parainfluenza virus 3] AHX22210.1 phosphoprotein[Human parainfluenza virus 3] AGW51105.1 phosphoprotein [Humanparainfluenza virus 3] AGT75251.1 phosphoprotein [Human parainfluenzavirus 3] AHX22362.1 phosphoprotein [Human parainfluenza virus 3]AHX22474.1 phosphoprotein [Human parainfluenza virus 3] AGW51217.1phosphoprotein [Human parainfluenza virus 3] AIG60038.1 phosphoprotein[Human parainfluenza virus 3] AHX22378.1 phosphoprotein [Humanparainfluenza virus 3] AGW51057.1 phosphoprotein [Human parainfluenzavirus 3] AGT75187.1 phosphoprotein [Human parainfluenza virus 3]AGW51233.1 phosphoprotein [Human parainfluenza virus 3] AHX22482.1phosphoprotein [Human parainfluenza virus 3] AGW51161.1 phosphoprotein[Human parainfluenza virus 3] AHX22306.1 phosphoprotein [Humanparainfluenza virus 3] AHX22162.1 phosphoprotein [Human parainfluenzavirus 3] ACJ70087.1 phosphoprotein [Human parainfluenza virus 3]AHX22466.1 phosphoprotein [Human parainfluenza virus 3] AHX22346.1phosphoprotein [Human parainfluenza virus 3] AGW51089.1 phosphoprotein[Human parainfluenza virus 3] AGW51073.1 phosphoprotein [Humanparainfluenza virus 3] AGW51185.1 phosphoprotein [Human parainfluenzavirus 3] AGW51065.1 phosphoprotein [Human parainfluenza virus 3]ABY47603.1 phosphoprotein [Human parainfluenza virus 3] AGW51049.1phosphoprotein [Human parainfluenza virus 3] AHX22330.1 phosphoprotein[Human parainfluenza virus 3] AGW51250.1 phosphoprotein [Humanparainfluenza virus 3] AGT75227.1 phosphoprotein [Human parainfluenzavirus 3] AGW51282.1 phosphoprotein [Human parainfluenza virus 3]AGW51209.1 phosphoprotein [Human parainfluenza virus 3] AGW51193.1phosphoprotein [Human parainfluenza virus 3] AGT75322.1 phosphoprotein[Human parainfluenza virus 3] AGT75219.1 phosphoprotein [Humanparainfluenza virus 3] AGW51258.1 phosphoprotein [Human parainfluenzavirus 3] AGW51041.1 phosphoprotein [Human parainfluenza virus 3]ACD99698.1 phosphoprotein [Human parainfluenza virus 3] AGW51266.1phosphoprotein [Human parainfluenza virus 3] AGT75179.1 phosphoprotein[Human parainfluenza virus 3] AHX22282.1 phosphoprotein [Humanparainfluenza virus 3] AGW51169.1 phosphoprotein [Human parainfluenzavirus 3] AGW51274.1 phosphoprotein [Human parainfluenza virus 3]AGW51201.1 phosphoprotein [Human parainfluenza virus 3] AGW51177.1RecName: Full = Phosphoprotein; Short = Protein P P06162.1 P protein[Human parainfluenza virus 3] AAA66818.1 phosphoprotein [Humanparainfluenza virus 3] AAA46866.1 phosphoprotein [Human parainfluenzavirus 3] BAA00031.1 polymerase-associated nucleocapsid phosphoproteinRRNZP5 (version 2) - parainfluenza virus type 3 [Human parainfluenzavirus 3] phosphoprotein [Human parainfluenza virus 3] AGT75171.1phosphoprotein [Human parainfluenza virus 3] BAA00921.1 D protein [Humanparainfluenza virus 3] NP_599250.1 D protein [Human parainfluenza virus3] AHX22377.1 D protein [Human parainfluenza virus 3] AHX22121.1 Dprotein [Human parainfluenza virus 3] AGT75297.1 D protein [Humanparainfluenza virus 3] AGW51136.1 D protein [Human parainfluenza virus3] AGW51242.1 D protein [Human parainfluenza virus 3] AGW51112.1 Dprotein [Human parainfluenza virus 3] AHX22497.1 D protein [Humanparainfluenza virus 3] AHX22145.1 D protein [Human parainfluenza virus3] AGT75202.1 D protein [Human parainfluenza virus 3] AHX22385.1 Dprotein [Human parainfluenza virus 3] AGW51216.1 D protein [Humanparainfluenza virus 3] AGT75281.1 D protein [Human parainfluenza virus3] AGT75194.1 D protein [Human parainfluenza virus 3] AHX22521.1 Dprotein [Human parainfluenza virus 3] AGW51120.1 D protein [Humanparainfluenza virus 3] AGT75313.1 D protein [Human parainfluenza virus3] AHX22249.1 D protein [Human parainfluenza virus 3] AHX22097.1 Dprotein [Human parainfluenza virus 3] AGW51144.1 D protein [Humanparainfluenza virus 3] AHX22089.1 D protein [Human parainfluenza virus3] AHX22225.1 D protein [Human parainfluenza virus 3] AHX22137.1 Dprotein [Human parainfluenza virus 3] AHX22065.1 D protein [Humanparainfluenza virus 3] AGW51224.1 D protein [Human parainfluenza virus3] AGT75210.1 D protein [Human parainfluenza virus 3] AHX22393.1 Dprotein [Human parainfluenza virus 3] AGT75258.1 D protein [Humanparainfluenza virus 3] AHX22345.1 D protein [Human parainfluenza virus3] AGT75250.1 D protein [Human parainfluenza virus 3] AHX22113.1 Dprotein [Human parainfluenza virus 3] AGW51232.1 D protein [Humanparainfluenza virus 3] AHX22057.1 D protein [Human parainfluenza virus3] AHX22209.1 D protein [Human parainfluenza virus 3] AGW51056.1 Dprotein [Human parainfluenza virus 3] AHX22161.1 D protein [Simian Agent10] ADR00402.1 D protein [Human parainfluenza virus 3] AHX22361.1 Dprotein [Human parainfluenza virus 3] AGW51281.1 D protein [Humanparainfluenza virus 3] AGW51184.1 D protein [Human parainfluenza virus3] AGW51160.1 D protein [Human parainfluenza virus 3] AHX22465.1 Dprotein [Human parainfluenza virus 3] AHX22329.1 D protein [Humanparainfluenza virus 3] AGW51064.1 D protein [Human parainfluenza virus3] AGW51040.1 D protein [Human parainfluenza virus 3] AGT75226.1 Dprotein [Human parainfluenza virus 3] AHX22425.1 D protein [Humanparainfluenza virus 3] AHX22305.1 D protein [Human parainfluenza virus3] AGW51249.1 D protein [Human parainfluenza virus 3] AHX22481.1 Dprotein [Human parainfluenza virus 3] AHX22281.1 D protein [Humanparainfluenza virus 3] AGW51048.1 D protein [Human parainfluenza virus3] AHX22297.1 D protein [Human parainfluenza virus 3] AGW51088.1 Dprotein [Human parainfluenza virus 3] AGT75305.1 D protein [Humanparainfluenza virus 3] AHX22185.1 D protein [Human parainfluenza virus3] AGW51104.1 D protein [Human parainfluenza virus 3] AHX22081.1 Dprotein [Human parainfluenza virus 3] AGW51192.1 D protein [Humanparainfluenza virus 3] AHX22489.1 D protein [Human parainfluenza virus3] AHX22441.1 D protein [Human parainfluenza virus 3] AHX22409.1 Dprotein [Human parainfluenza virus 3] AHX22369.1 D protein [Humanparainfluenza virus 3] AHX22321.1 D protein [Human parainfluenza virus3] AHX22073.1 D protein [Human parainfluenza virus 3] AGW51152.1 Dprotein [Human parainfluenza virus 3] AGW51072.1 D protein [Humanparainfluenza virus 3] AGT75321.1 D protein [Human parainfluenza virus3] AHX22257.1 D protein [Human parainfluenza virus 3] AHX22129.1 Dprotein [Human parainfluenza virus 3] AHX22417.1 D protein [Humanparainfluenza virus 3] AGT75218.1 D protein [Human parainfluenza virus3] AHX22265.1 D protein [Human parainfluenza virus 3] AGT75178.1 Dprotein [Human parainfluenza virus 3] AHX22433.1 D protein [Humanparainfluenza virus 3] AGW51273.1 D protein [Human parainfluenza virus3] AGW51208.1 D protein [Human parainfluenza virus 3] AGT75170.1 Dprotein [Human parainfluenza virus 3] AGT75162.1 D protein [Humanparainfluenza virus 3] AGW51257.1 D protein [Human parainfluenza virus3] AGW51200.1 D protein [Human parainfluenza virus 3] AGW51176.1 Dprotein [Human parainfluenza virus 3] AGT75186.1 D protein [Humanparainfluenza virus 3] AGW51265.1 D protein [Human parainfluenza virus3] AGW51168.1

TABLE 8 Signal Peptides SEQ Description Sequence ID NO: HuIgG_(k) signalMETPAQLLFLLL 15 peptide LWLPDTTG IgE heavy chain MDWTWILFLVAA 16epsilon -1 signal ATRVHS peptide Japanese MLGSNSGQRVVF 17encephalitis PRM TILLLLVAPAYS signal sequence VSVg protein MKCLLYLAFLFI18 signal sequence GVNCA Japanese MWLVSLAIVTAC 19 encephalitis JEV AGAsignal sequence

TABLE 9 hMPV/PIV Cotton Rat Challenge Study Design Group n Test Article[conc]/μg Route Challenge 1 5 Placebo n/a IM hMPV/A2 2 5 hMPV vaccinemRNA 30 IM hMPV/A2 3 5 hMPV vaccine mRNA 15 IM hMPV/A2 4 5 hMPV vaccinemRNA 10 IM hMPV/A2 5 5 hMPV/PIV3 vaccine 30 IM hMPV/A2 mRNA (15/15) 6 5FI-hMPV n/a IM hMPV/A2 7 5 Placebo n/a IM PIV3 8 5 PIV3 vaccine mRNA 30IM PIV3 9 5 PIV3 vaccine mRNA 15 IM PIV3 10 5 PIV3 vaccine mRNA 10 IMPIV3 11 5 hMPV/PIV3 vaccine 30 IM PIV3 mRNA (15/15) 12 5 FI-PIV3 n/a IMPIV3 60

TABLE 10 SEQ ID Strain Nucleic Acid Sequence NO:Betacoronavirus Nucleic Acid Sequence gb|KJ156934.1|:ATGATACACTCAGTGTTTCTACTGATGTTCTTGTTAACACC 20 21405-25466 MiddleTACAGAAAGTTACGTTGATGTAGGGCCAGATTCTGTTAAG East respiratoryTCTGCTTGTATTGAGGTTGATATACAACAGACCTTCTTTGA syndrome coronavirusTAAAACTTGGCCTAGGCCAATTGATGTTTCTAAGGCTGAC isolateGGTATTATATACCCTCAAGGCCGTACATATTCTAACATAA Riyadh_14_2013,CTATCACTTATCAAGGTCTTTTTCCCTATCAGGGAGACCAT spike proteinGGTGATATGTATGTTTACTCTGCAGGACATGCTACAGGCA (nucleotide)CAACTCCACAAAAGTTGTTTGTAGCTAACTATTCTCAGGACGTCAAACAGTTTGCTAATGGGTTTGTCGTCCGTATAGGAGCAGCTGCCAATTCCACTGGCACTGTTATTATTAGCCCATCTACCAGCGCTACTATACGAAAAATTTACCCTGCTTTTATGCTGGGTTCTTCAGTTGGTAATTTCTCAGATGGTAAAATGGGCCGCTTCTTCAATCATACTCTAGTTCTTTTGCCCGATGGATGTGGCACTTTACTTAGAGCTTTTTATTGTATTCTAGAGCCTCGCTCTGGAAATCATTGTCCTGCTGGCAATTCCTATACTTCTTTTGCCACTTATCACACTCCTGCAACAGATTGTTCTGATGGCAATTACAATCGTAATGCCAGTCTGAACTCTTTTAAGGAGTATTTTAATTTACGTAACTGCACCTTTATGTACACTTATAACATTACCGAAGATGAGATTTTAGAGTGGTTTGGCATTACACAAACTGCTCAAGGTGTTCACCTCTTCTCATCTCGGTATGTTGATTTGTACGGCGGCAATATGTTTCAATTTGCCACCTTGCCTGTTTATGATACTATTAAGTATTATTCTATCATTCCTCACAGTATTCGTTCTATCCAAAGTGATAGAAAAGCTTGGGCTGCCTTCTACGTATATAAACTTCAACCGTTAACTTTCCTGTTGGATTTTTCTGTTGATGGTTATATACGCAGAGCTATAGACTGTGGTTTTAATGATTTGTCACAACTCCACTGCTCATATGAATCCTTCGATGTTGAATCTGGAGTTTATTCAGTTTCGTCTTTCGAAGCAAAACCTTCTGGCTCAGTTGTGGAACAGGCTGAAGGTGTTGAATGTGATTTTTCACCTCTTCTGTCTGGCACACCTCCTCAGGTTTATAATTTCAAGCGTTTGGTTTTTACCAATTGCAATTATAATCTTACCAAATTGCTTTCACTTTTTTCTGTGAATGATTTTACTTGTAGTCAAATATCTCCAGCAGCAATTGCTAGCAACTGTTATTCTTCACTGATTTTGGATTATTTTTCATACCCACTTAGTATGAAATCCGATCTCAGTGTTAGTTCTGCTGGTCCAATATCCCAGTTTAATTATAAACAGTCCTTTTCTAATCCCACATGTTTGATCTTAGCGACTGTTCCTCATAACCTTACTACTATTACTAAGCCTCTTAAGTACAGCTATATTAACAAGTGCTCTCGTCTTCTTTCTGATGATCGTACTGAAGTACCTCAGTTAGTGAACGCTAATCAATACTCACCCTGTGTATCCATTGTCCCATCCACTGTGTGGGAAGACGGTGATTATTATAGGAAACAACTATCTCCACTTGAAGGTGGTGGCTGGCTTGTTGCTAGTGGCTCAACTGTTGCCATGACTGAGCAATTACAGATGGGCTTTGGTATTACAGTTCAATATGGTACAGACACCAATAGTGTTTGCCCCAAGCTTGAATTTGCTAATGACACAAAAATTGCCTCTCAATTAGGCAATTGCGTGGAATATTCCCTCTATGGTGTTTCGGGCCGTGGTGTTTTTCAGAATTGCACAGCTGTAGGTGTTCGACAGCAGCGCTTTGTTTATGATGCGTACCAGAATTTAGTTGGCTATTATTCTGATGATGGCAACTACTACTGTCTGCGTGCTTGTGTTAGTGTTCCTGTTTCTGTCATCTATGATAAAGAAACTAAAACCCACGCTACTCTATTTGGTAGTGTTGCATGTGAACACATTTCTTCTACCATGTCTCAATACTCCCGTTCTACGCGATCAATGCTTAAACGGCGAGATTCTACATATGGCCCCCTTCAGACACCTGTTGGTTGTGTCCTAGGACTTGTTAATTCCTCTTTGTTCGTAGAGGACTGCAAGTTGCCTCTCGGTCAATCTCTCTGTGCTCTTCCTGACACACCTAGTACTCTCACACCTCGCAGTGTGCGCTCTGTGCCAGGTGAAATGCGCTTGGCATCCATTGCTTTTAATCATCCCATTCAGGTTGATCAACTTAATAGTAGTTATTTTAAATTAAGTATACCCACTAATTTTTCCTTTGGTGTGACTCAGGAGTACATTCAGACAACCATTCAGAAAGTTACTGTTGATTGTAAACAGTACGTTTGCAATGGTTTCCAGAAGTGTGAGCAATTACTGCGCGAGTATGGCCAGTTTTGTTCCAAAATAAACCAGGCTCTCCATGGTGCCAATTTACGCCAGGATGATTCTGTACGTAATTTGTTTGCGAGCGTGAAAAGCTCTCAATCATCTCCTATCATACCAGGTTTTGGAGGTGACTTTAATTTGACACTTCTAGAACCTGTTTCTATATCTACTGGCAGTCGTAGTGCACGTAGTGCTATTGAGGATTTGCTATTTGACAAAGTCACTATAGCTGATCCTGGTTATATGCAAGGTTACGATGATTGTATGCAGCAAGGTCCAGCATCAGCTCGTGATCTTATTTGTGCTCAATATGTGGCTGGTTATAAAGTATTACCTCCTCTTATGGATGTTAATATGGAAGCCGCGTATACTTCATCTTTGCTTGGCAGCATAGCAGGTGTTGGCTGGACTGCTGGCTTATCCTCCTTTGCTGCTATTCCATTTGCACAGAGTATYTTTTATAGGTTAAACGGTGTTGGCATTACTCAACAGGTTCTTTCAGAGAACCAAAAGCTTATTGCCAATAAGTTTAATCAGGCTCTGGGAGCTATGCAAACAGGCTTCACTACAACTAATGAAGCTTTTCGGAAGGTTCAGGATGCTGTGAACAACAATGCACAGGCTCTATCCAAATTAGCTAGCGAGCTATCTAATACTTTTGGTGCTATTTCCGCCTCTATTGGAGACATCATACAACGTCTTGATGTTCTCGAACAGGACGCCCAAATAGACAGACTTATTAATGGCCGTTTGACAACACTAAATGCTTTTGTTGCACAGCAGCTTGTTCGTTCCGAATCAGCTGCTCTTTCCGCTCAATTGGCTAAAGATAAAGTCAATGAGTGTGTCAAGGCACAATCCAAGCGTTCTGGATTTTGCGGTCAAGGCACACATATAGTGTCCTTTGTTGTAAATGCCCCTAATGGCCTTTACTTTATGCATGTTGGTTATTACCCTAGCAACCACATTGAGGTTGTTTCTGCTTATGGTCTTTGCGATGCAGCTAACCCTACTAATTGTATAGCCCCTGTTAATGGCTACTTTATTAAAACTAATAACACTAGGATTGTTGATGAGTGGTCATATACTGGCTCGTCCTTCTATGCACCTGAGCCCATCACCTCTCTTAATACTAAGTATGTTGCACCACAGGTGACATACCAAAACATTTCTACTAACCTCCCTCCTCCTCTTCTCGGCAATTCCACCGGGATTGACTTCCAAGATGAGTTGGATGAGTTTTTCAAAAATGTTAGCACCAGTATACCTAATTTTGGTTCTCTAACACAGATTAATACTACATTACTCGATCTTACCTACGAGATGTTGTCTCTTCAACAAGTTGTTAAAGCCCTTAATGAGTCTTACATAGACCTTAAAGAGCTTGGCAATTATACTTATTACAACAAATGGCCGTGGTACATTTGGCTTGGTTTCATTGCTGGGCTTGTTGCCTTAGCTCTATGCGTCTTCTTCATACTGTGCTGCACTGGTTGTGGCACAAACTGTATGGGAAAACTTAAGTGTAATCGTTGTTGTGATAGATACGAGGAATACGACCTCGAGCCGCATAAGGTTCATGTTCA CTAA MERS S FLATGATACACTCAGTGTTTCTACTGATGTTCTTGTTAACACC 21 SPIKE 2cEMC/2012TACAGAAAGTTACGTTGATGTAGGGCCAGATTCTGTTAAG (XBaI changeTCTGCTTGTATTGAGGTTGATATACAACAGACTTTCTTTGA (T to G)) TAAAACTTGGCCTAGGCCAATTGATGTTTCTAAGGCTGAC (nucleotide)GGTATTATATACCCTCAAGGCCGTACATATTCTAACATAACTATCACTTATCAAGGTCTTTTTCCCTATCAGGGAGACCATGGTGATATGTATGTTTACTCTGCAGGACATGCTACAGGCACAACTCCACAAAAGTTGTTTGTAGCTAACTATTCTCAGGACGTCAAACAGTTTGCTAATGGGTTTGTCGTCCGTATAGGAGCAGCTGCCAATTCCACTGGCACTGTTATTATTAGCCCATCTACCAGCGCTACTATACGAAAAATTTACCCTGCTTTTATGCTGGGTTCTTCAGTTGGTAATTTCTCAGATGGTAAAATGGGCCGCTTCTTCAATCATACTCTAGTTCTTTTGCCCGATGGATGTGGCACTTTACTTAGAGCTTTTTATTGTATTCTGGAGCCTCGCTCTGGAAATCATTGTCCTGCTGGCAATTCCTATACTTCTTTTGCCACTTATCACACTCCTGCAACAGATTGTTCTGATGGCAATTACAATCGTAATGCCAGTCTGAACTCTTTTAAGGAGTATTTTAATTTACGTAACTGCACCTTTATGTACACTTATAACATTACCGAAGATGAGATTTTAGAGTGGTTTGGCATTACACAAACTGCTCAAGGTGTTCACCTCTTCTCATCTCGGTATGTTGATTTGTACGGCGGCAATATGTTTCAATTTGCCACCTTGCCTGTTTATGATACTATTAAGTATTATTCTATCATTCCTCACAGTATTCGTTCTATCCAAAGTGATAGAAAAGCTTGGGCTGCCTTCTACGTATATAAACTTCAACCGTTAACTTTCCTGTTGGATTTTTCTGTTGATGGTTATATACGCAGAGCTATAGACTGTGGTTTTAATGATTTGTCACAACTCCACTGCTCATATGAATCCTTCGATGTTGAATCTGGAGTTTATTCAGTTTCGTCTTTCGAAGCAAAACCTTCTGGCTCAGTTGTGGAACAGGCTGAAGGTGTTGAATGTGATTTTTCACCTCTTCTGTCTGGCACACCTCCTCAGGTTTATAATTTCAAGCGTTTGGTTTTTACCAATTGCAATTATAATCTTACCAAATTGCTTTCACTTTTTTCTGTGAATGATTTTACTTGTAGTCAAATATCTCCAGCAGCAATTGCTAGCAACTGTTATTCTTCACTGATTTTGGATTACTTTTCATACCCACTTAGTATGAAATCCGATCTCAGTGTTAGTTCTGCTGGTCCAATATCCCAGTTTAATTATAAACAGTCCTTTTCTAATCCCACATGTTTGATTTTAGCGACTGTTCCTCATAACCTTACTACTATTACTAAGCCTCTTAAGTACAGCTATATTAACAAGTGCTCTCGTCTTCTTTCTGATGATCGTACTGAAGTACCTCAGTTAGTGAACGCTAATCAATACTCACCCTGTGTATCCATTGTCCCATCCACTGTGTGGGAAGACGGTGATTATTATAGGAAACAACTATCTCCACTTGAAGGTGGTGGCTGGCTTGTTGCTAGTGGCTCAACTGTTGCCATGACTGAGCAATTACAGATGGGCTTTGGTATTACAGTTCAATATGGTACAGACACCAATAGTGTTTGCCCCAAGCTTGAATTTGCTAATGACACAAAAATTGCCTCTCAATTAGGCAATTGCGTGGAATATTCCCTCTATGGTGTTTCGGGCCGTGGTGTTTTTCAGAATTGCACAGCTGTAGGTGTTCGACAGCAGCGCTTTGTTTATGATGCGTACCAGAATTTAGTTGGCTATTATTCTGATGATGGCAACTACTACTGTTTGCGTGCTTGTGTTAGTGTTCCTGTTTCTGTCATCTATGATAAAGAAACTAAAACCCACGCTACTCTATTTGGTAGTGTTGCATGTGAACACATTTCTTCTACCATGTCTCAATACTCCCGTTCTACGCGATCAATGCTTAAACGGCGAGATTCTACATATGGCCCCCTTCAGACACCTGTTGGTTGTGTCCTAGGACTTGTTAATTCCTCTTTGTTCGTAGAGGACTGCAAGTTGCCTCTTGGTCAATCTCTCTGTGCTCTTCCTGACACACCTAGTACTCTCACACCTCGCAGTGTGCGCTCTGTTCCAGGTGAAATGCGCTTGGCATCCATTGCTTTTAATCATCCTATTCAGGTTGATCAACTTAATAGTAGTTATTTTAAATTAAGTATACCCACTAATTTTTCCTTTGGTGTGACTCAGGAGTACATTCAGACAACCATTCAGAAAGTTACTGTTGATTGTAAACAGTACGTTTGCAATGGTTTCCAGAAGTGTGAGCAATTACTGCGCGAGTATGGCCAGTTTTGTTCCAAAATAAACCAGGCTCTCCATGGTGCCAATTTACGCCAGGATGATTCTGTACGTAATTTGTTTGCGAGCGTGAAAAGCTCTCAATCATCTCCTATCATACCAGGTTTTGGAGGTGACTTTAATTTGACACTTCTGGAACCTGTTTCTATATCTACTGGCAGTCGTAGTGCACGTAGTGCTATTGAGGATTTGCTATTTGACAAAGTCACTATAGCTGATCCTGGTTATATGCAAGGTTACGATGATTGCATGCAGCAAGGTCCAGCATCAGCTCGTGATCTTATTTGTGCTCAATATGTGGCTGGTTACAAAGTATTACCTCCTCTTATGGATGTTAATATGGAAGCCGCGTATACTTCATCTTTGCTTGGCAGCATAGCAGGTGTTGGCTGGACTGCTGGCTTATCCTCCTTTGCTGCTATTCCATTTGCACAGAGTATCTTTTATAGGTTAAACGGTGTTGGCATTACTCAACAGGTTCTTTCAGAGAACCAAAAGCTTATTGCCAATAAGTTTAATCAGGCTCTGGGAGCTATGCAAACAGGCTTCACTACAACTAATGAAGCTTTTCAGAAGGTTCAGGATGCTGTGAACAACAATGCACAGGCTCTATCCAAATTAGCTAGCGAGCTATCTAATACTTTTGGTGCTATTTCCGCCTCTATTGGAGACATCATACAACGTCTTGATGTTCTCGAACAGGACGCCCAAATAGACAGACTTATTAATGGCCGTTTGACAACACTAAATGCTTTTGTTGCACAGCAGCTTGTTCGTTCCGAATCAGCTGCTCTTTCCGCTCAATTGGCTAAAGATAAAGTCAATGAGTGTGTCAAGGCACAATCCAAGCGTTCTGGATTTTGCGGTCAAGGCACACATATAGTGTCCTTTGTTGTAAATGCCCCTAATGGCCTTTACTTCATGCATGTTGGTTATTACCCTAGCAACCACATTGAGGTTGTTTCTGCTTATGGTCTTTGCGATGCAGCTAACCCTACTAATTGTATAGCCCCTGTTAATGGCTACTTTATTAAAACTAATAACACTAGGATTGTTGATGAGTGGTCATATACTGGCTCGTCCTTCTATGCACCTGAGCCCATTACCTCCCTTAATACTAAGTATGTTGCACCACAGGTGACATACCAAAACATTTCTACTAACCTCCCTCCTCCTCTTCTCGGCAATTCCACCGGGATTGACTTCCAAGATGAGTTGGATGAGTTTTTCAAAAATGTTAGCACCAGTATACCTAATTTTGGTTCCCTAACACAGATTAATACTACATTACTCGATCTTACCTACGAGATGTTGTCTCTTCAACAAGTTGTTAAAGCCCTTAATGAGTCTTACATAGACCTTAAAGAGCTTGGCAATTATACTTATTACAACAAATGGCCGTGGTACATTTGGCTTGGTTTCATTGCTGGGCTTGTTGCCTTAGCTCTATGCGTCTTCTTCATACTGTGCTGCACTGGTTGTGGCACAAACTGTATGGGAAAACTTAAGTGTAATCGTTGTTGTGATAGATACGAGGAATACGACCTCGAGCCGCATAAGGTTCATGTTCAC TAA Novel_MERS_S2_sub-ATGATCCACTCCGTGTTCCTCCTCATGTTCCTGTTGACCCC 22 unit_trimericCACTGAGTCAGACTGCAAGCTCCCGCTGGGACAGTCCCTG vaccineTGTGCGCTGCCTGACACTCCTAGCACTCTGACCCCACGCTC (nucleotide)CGTGCGGTCGGTGCCTGGCGAAATGCGGCTGGCCTCCATCGCCTTCAATCACCCAATCCAAGTGGATCAGCTGAATAGCTCGTATTTCAAGCTGTCCATCCCCACGAACTTCTCGTTCGGGGTCACCCAGGAGTACATCCAGACCACAATTCAGAAGGTCACCGTCGATTGCAAGCAATACGTGTGCAACGGCTTCCAGAAGTGCGAGCAGCTGCTGAGAGAATACGGGCAGTTTTGCAGCAAGATCAACCAGGCGCTGCATGGAGCTAACTTGCGCCAGGACGACTCCGTGCGCAACCTCTTTGCCTCTGTGAAGTCATCCCAGTCCTCCCCAATCATCCCGGGATTCGGAGGGGACTTCAACCTGACCCTCCTGGAGCCCGTGTCGATCAGCACCGGTAGCAGATCGGCGCGCTCAGCCATTGAAGATCTTCTGTTCGACAAGGTCACCATCGCCGATCCGGGCTACATGCAGGGATACGACGACTGTATGCAGCAGGGACCAGCCTCCGCGAGGGACCTCATCTGCGCGCAATACGTGGCCGGGTACAAAGTGCTGCCTCCTCTGATGGATGTGAACATGGAGGCCGCTTATACTTCGTCCCTGCTCGGCTCTATCGCCGGCGTGGGGTGGACCGCCGGCCTGTCCTCCTTCGCCGCTATCCCCTTTGCACAATCCATTTTCTACCGGCTCAACGGCGTGGGCATTACTCAACAAGTCCTGTCGGAGAACCAGAAGTTGATCGCAAACAAGTTCAATCAGGCCCTGGGGGCCATGCAGACTGGATTCACTACGACTAACGAAGCGTTCCAGAAGGTCCAGGACGCTGTGAACAACAACGCCCAGGCGCTCTCAAAGCTGGCCTCCGAACTCAGCAACACCTTCGGAGCCATCAGCGCATCGATCGGTGACATAATTCAGCGGCTGGACGTGCTGGAGCAGGACGCCCAGATCGACCGCCTCATCAACGGACGGCTGACCACCTTGAATGCCTTCGTGGCACAACAGCTGGTCCGGAGCGAATCAGCGGCACTTTCCGCCCAACTCGCCAAGGACAAAGTCAACGAATGCGTGAAGGCCCAGTCCAAGAGGTCCGGTTTCTGCGGTCAAGGAACCCATATTGTGTCCTTCGTCGTGAACGCGCCCAACGGTCTGTACTTTATGCACGTCGGCTACTACCCGAGCAATCATATCGAAGTGGTGTCCGCCTACGGCCTGTGCGATGCCGCTAACCCCACTAACTGTATTGCCCCTGTGAACGGATATTTTATTAAGACCAACAACACCCGCATTGTGGACGAATGGTCATACACCGGTTCGTCCTTCTACGCGCCCGAGCCCATCACTTCACTGAACACCAAATACGTGGCTCCGCAAGTGACCTACCAGAACATCTCCACCAATTTGCCGCCGCCGCTGCTCGGAAACAGCACCGGAATTGATTTCCAAGATGAACTGGACGAATTCTTCAAGAACGTGTCCACTTCCATTCCCAACTTCGGAAGCCTGACACAGATCAACACCACCCTTCTCGACCTGACCTACGAGATGCTGAGCCTTCAACAAGTGGTCAAGGCCCTGAACGAGAGCTACATCGACCTGAAGGAGCTGGGCAACTATACCTACTACAACAAGTGGCCGGACAAGATTGAGGAGATTCTGTCGAAAATCTACCACATTGAAAACGAGATCGCCAGAATCAAGAAGCTTATCGGCGA AGCC MERS_S0_Full-ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTG 23 length SpikeGCTGCCTGATACCACCGGCAGCTATGTGGACGTGGGCCCC proteinGATAGCGTGAAGTCCGCCTGTATCGAAGTGGACATCCAGC (nucleotide,AGACCTTTTTCGACAAGACCTGGCCCAGACCCATCGACGT codonGTCCAAGGCCGACGGCATCATCTATCCACAAGGCCGGACC optimized)TACAGCAACATCACCATTACCTACCAGGGCCTGTTCCCATATCAAGGCGACCACGGCGATATGTACGTGTACTCTGCCGGCCACGCCACCGGCACCACACCCCAGAAACTGTTCGTGGCCAACTACAGCCAGGACGTGAAGCAGTTCGCCAACGGCTTCGTCGTGCGGATTGGCGCCGCTGCCAATAGCACCGGCACAGTGATCATCAGCCCCAGCACCAGCGCCACCATCCGGAAGATCTACCCCGCCTTCATGCTGGGCAGCTCCGTGGGCAATTTCAGCGACGGCAAGATGGGCCGGTTCTTCAACCACACCCTGGTGCTGCTGCCCGATGGCTGTGGCACACTGCTGAGAGCCTTCTACTGCATCCTGGAACCCAGAAGCGGCAACCACTGCCCTGCCGGCAATAGCTACACCAGCTTCGCCACCTACCACACACCCGCCACCGATTGCTCCGACGGCAACTACAACCGGAACGCCAGCCTGAACAGCTTCAAAGAGTACTTCAACCTGCGGAACTGCACCTTCATGTACACCTACAATATCACCGAGGACGAGATCCTGGAATGGTTCGGCATCACCCAGACCGCCCAGGGCGTGCACCTGTTCAGCAGCAGATACGTGGACCTGTACGGCGGCAACATGTTCCAGTTTGCCACCCTGCCCGTGTACGACACCATCAAGTACTACAGCATCATCCCCCACAGCATCCGGTCCATCCAGAGCGACAGAAAAGCCTGGGCCGCCTTCTACGTGTACAAGCTGCAGCCCCTGACCTTCCTGCTGGACTTCAGCGTGGACGGCTACATCAGACGGGCCATCGACTGCGGCTTCAACGACCTGAGCCAGCTGCACTGCTCCTACGAGAGCTTCGACGTGGAAAGCGGCGTGTACAGCGTGTCCAGCTTCGAGGCCAAGCCTAGCGGCAGCGTGGTGGAACAGGCTGAGGGCGTGGAATGCGACTTCAGCCCTCTGCTGAGCGGCACCCCTCCCCAGGTGTACAACTTCAAGCGGCTGGTGTTCACCAACTGCAATTACAACCTGACCAAGCTGCTGAGCCTGTTCTCCGTGAACGACTTCACCTGTAGCCAGATCAGCCCTGCCGCCATTGCCAGCAACTGCTACAGCAGCCTGATCCTGGACTACTTCAGCTACCCCCTGAGCATGAAGTCCGATCTGAGCGTGTCCTCCGCCGGACCCATCAGCCAGTTCAACTACAAGCAGAGCTTCAGCAACCCTACCTGCCTGATTCTGGCCACCGTGCCCCACAATCTGACCACCATCACCAAGCCCCTGAAGTACAGCTACATCAACAAGTGCAGCAGACTGCTGTCCGACGACCGGACCGAAGTGCCCCAGCTCGTGAACGCCAACCAGTACAGCCCCTGCGTGTCCATCGTGCCCAGCACCGTGTGGGAGGACGGCGACTACTACAGAAAGCAGCTGAGCCCCCTGGAAGGCGGCGGATGGCTGGTGGCTTCTGGAAGCACAGTGGCCATGACCGAGCAGCTGCAGATGGGCTTTGGCATCACCGTGCAGTACGGCACCGACACCAACAGCGTGTGCCCCAAGCTGGAATTCGCCAATGACACCAAGATCGCCAGCCAGCTGGGAAACTGCGTGGAATACTCCCTGTATGGCGTGTCCGGACGGGGCGTGTTCCAGAATTGCACAGCAGTGGGAGTGCGGCAGCAGAGATTCGTGTACGATGCCTACCAGAACCTCGTGGGCTACTACAGCGACGACGGCAATTACTACTGCCTGCGGGCCTGTGTGTCCGTGCCCGTGTCCGTGATCTACGACAAAGAGACAAAGACCCACGCCACACTGTTCGGCTCCGTGGCCTGCGAGCACATCAGCTCCACCATGAGCCAGTACTCCCGCTCCACCCGGTCCATGCTGAAGCGGAGAGATAGCACCTACGGCCCCCTGCAGACACCTGTGGGATGTGTGCTGGGCCTCGTGAACAGCTCCCTGTTTGTGGAAGATTGCAAGCTGCCCCTGGGCCAGAGCCTGTGTGCCCTGCCAGATACCCCTAGCACCCTGACCCCTAGAAGCGTGCGCTCTGTGCCCGGCGAAATGCGGCTGGCCTCTATCGCCTTCAATCACCCCATCCAGGTGGACCAGCTGAACTCCAGCTACTTCAAGCTGAGCATCCCCACCAACTTCAGCTTCGGCGTGACCCAGGAGTACATCCAGACCACAATCCAGAAAGTGACCGTGGACTGCAAGCAGTACGTGTGCAACGGCTTTCAGAAGTGCGAACAGCTGCTGCGCGAGTACGGCCAGTTCTGCAGCAAGATCAACCAGGCCCTGCACGGCGCCAACCTGAGACAGGATGACAGCGTGCGGAACCTGTTCGCCAGCGTGAAAAGCAGCCAGTCCAGCCCCATCATCCCTGGCTTCGGCGGCGACTTTAACCTGACCCTGCTGGAACCTGTGTCCATCAGCACCGGCTCCAGAAGCGCCAGATCCGCCATCGAGGACCTGCTGTTCGACAAAGTGACCATTGCCGACCCCGGCTACATGCAGGGCTACGACGATTGCATGCAGCAGGGCCCAGCCAGCGCCAGGGATCTGATCTGTGCCCAGTATGTGGCCGGCTACAAGGTGCTGCCCCCCCTGATGGACGTGAACATGGAAGCCGCCTACACCTCCAGCCTGCTGGGCTCTATTGCTGGCGTGGGATGGACAGCCGGCCTGTCTAGCTTTGCCGCCATCCCTTTCGCCCAGAGCATCTTCTACCGGCTGAACGGCGTGGGCATCACACAACAGGTGCTGAGCGAGAACCAGAAGCTGATCGCCAACAAGTTTAACCAGGCACTGGGCGCCATGCAGACCGGCTTCACCACCACCAACGAGGCCTTCAGAAAGGTGCAGGACGCCGTGAACAACAACGCCCAGGCTCTGAGCAAGCTGGCCTCCGAGCTGAGCAATACCTTCGGCGCCATCAGCGCCTCCATCGGCGACATCATCCAGCGGCTGGACGTGCTGGAACAGGACGCCCAGATCGACCGGCTGATCAACGGCAGACTGACCACCCTGAACGCCTTCGTGGCACAGCAGCTCGTGCGGAGCGAATCTGCCGCTCTGTCTGCTCAGCTGGCCAAGGACAAAGTGAACGAGTGCGTGAAGGCCCAGTCCAAGCGGAGCGGCTTTTGTGGCCAGGGCACCCACATCGTGTCCTTCGTCGTGAATGCCCCCAACGGCCTGTACTTTATGCACGTGGGCTATTACCCCAGCAACCACATCGAGGTGGTGTCCGCCTATGGCCTGTGCGACGCCGCCAATCCTACCAACTGTATCGCCCCCGTGAACGGCTACTTCATCAAGACCAACAACACCCGGATCGTGGACGAGTGGTCCTACACAGGCAGCAGCTTCTACGCCCCCGAGCCCATCACCTCCCTGAACACCAAATACGTGGCCCCCCAAGTGACATACCAGAACATCTCCACCAACCTGCCCCCTCCACTGCTGGGAAATTCCACCGGCATCGACTTCCAGGACGAGCTGGACGAGTTCTTCAAGAACGTGTCCACCTCCATCCCCAACTTCGGCAGCCTGACCCAGATCAACACCACTCTGCTGGACCTGACCTACGAGATGCTGTCCCTGCAACAGGTCGTGAAAGCCCTGAACGAGAGCTACATCGACCTGAAAGAGCTGGGGAACTACACCTACTACAACAAGTGGCCTTGGTACATTTGGCTGGGCTTTATCGCCGGCCTGGTGGCCCTGGCCCTGTGCGTGTTCTTCATCCTGTGCTGCACCGGCTGCGGCACCAATTGCATGGGCAAGCTGAAATGCAACCGGTGCTGCGACAGATACGAGGAAT ACGACCTGGAACCTCACAAAGTGCATGTGCACBetacoronavirus mRNA Sequences gb|KJ156934.1|:AUGAUACACUCAGUGUUUCUACUGAUGUUCUUGUUAAC 65 21405-25466ACCUACAGAAAGUUACGUUGAUGUAGGGCCAGAUUCUG Middle EastUUAAGUCUGCUUGUAUUGAGGUUGAUAUACAACAGACC respiratoryUUCUUUGAUAAAACUUGGCCUAGGCCAAUUGAUGUUUC syndromeUAAGGCUGACGGUAUUAUAUACCCUCAAGGCCGUACAU coronavirusAUUCUAACAUAACUAUCACUUAUCAAGGUCUUUUUCCCU isolateAUCAGGGAGACCAUGGUGAUAUGUAUGUUUACUCUGCA Riyadh_14_2013,GGACAUGCUACAGGCACAACUCCACAAAAGUUGUUUGU spike proteinAGCUAACUAUUCUCAGGACGUCAAACAGUUUGCUAAUG (nucleotide)GGUUUGUCGUCCGUAUAGGAGCAGCUGCCAAUUCCACUGGCACUGUUAUUAUUAGCCCAUCUACCAGCGCUACUAUACGAAAAAUUUACCCUGCUUUUAUGCUGGGUUCUUCAGUUGGUAAUUUCUCAGAUGGUAAAAUGGGCCGCUUCUUCAAUCAUACUCUAGUUCUUUUGCCCGAUGGAUGUGGCACUUUACUUAGAGCUUUUUAUUGUAUUCUAGAGCCUCGCUCUGGAAAUCAUUGUCCUGCUGGCAAUUCCUAUACUUCUUUUGCCACUUAUCACACUCCUGCAACAGAUUGUUCUGAUGGCAAUUACAAUCGUAAUGCCAGUCUGAACUCUUUUAAGGAGUAUUUUAAUUUACGUAACUGCACCUUUAUGUACACUUAUAACAUUACCGAAGAUGAGAUUUUAGAGUGGUUUGGCAUUACACAAACUGCUCAAGGUGUUCACCUCUUCUCAUCUCGGUAUGUUGAUUUGUACGGCGGCAAUAUGUUUCAAUUUGCCACCUUGCCUGUUUAUGAUACUAUUAAGUAUUAUUCUAUCAUUCCUCACAGUAUUCGUUCUAUCCAAAGUGAUAGAAAAGCUUGGGCUGCCUUCUACGUAUAUAAACUUCAACCGUUAACUUUCCUGUUGGAUUUUUCUGUUGAUGGUUAUAUACGCAGAGCUAUAGACUGUGGUUUUAAUGAUUUGUCACAACUCCACUGCUCAUAUGAAUCCUUCGAUGUUGAAUCUGGAGUUUAUUCAGUUUCGUCUUUCGAAGCAAAACCUUCUGGCUCAGUUGUGGAACAGGCUGAAGGUGUUGAAUGUGAUUUUUCACCUCUUCUGUCUGGCACACCUCCUCAGGUUUAUAAUUUCAAGCGUUUGGUUUUUACCAAUUGCAAUUAUAAUCUUACCAAAUUGCUUUCACUUUUUUCUGUGAAUGAUUUUACUUGUAGUCAAAUAUCUCCAGCAGCAAUUGCUAGCAACUGUUAUUCUUCACUGAUUUUGGAUUAUUUUUCAUACCCACUUAGUAUGAAAUCCGAUCUCAGUGUUAGUUCUGCUGGUCCAAUAUCCCAGUUUAAUUAUAAACAGUCCUUUUCUAAUCCCACAUGUUUGAUCUUAGCGACUGUUCCUCAUAACCUUACUACUAUUACUAAGCCUCUUAAGUACAGCUAUAUUAACAAGUGCUCUCGUCUUCUUUCUGAUGAUCGUACUGAAGUACCUCAGUUAGUGAACGCUAAUCAAUACUCACCCUGUGUAUCCAUUGUCCCAUCCACUGUGUGGGAAGACGGUGAUUAUUAUAGGAAACAACUAUCUCCACUUGAAGGUGGUGGCUGGCUUGUUGCUAGUGGCUCAACUGUUGCCAUGACUGAGCAAUUACAGAUGGGCUUUGGUAUUACAGUUCAAUAUGGUACAGACACCAAUAGUGUUUGCCCCAAGCUUGAAUUUGCUAAUGACACAAAAAUUGCCUCUCAAUUAGGCAAUUGCGUGGAAUAUUCCCUCUAUGGUGUUUCGGGCCGUGGUGUUUUUCAGAAUUGCACAGCUGUAGGUGUUCGACAGCAGCGCUUUGUUUAUGAUGCGUACCAGAAUUUAGUUGGCUAUUAUUCUGAUGAUGGCAACUACUACUGUCUGCGUGCUUGUGUUAGUGUUCCUGUUUCUGUCAUCUAUGAUAAAGAAACUAAAACCCACGCUACUCUAUUUGGUAGUGUUGCAUGUGAACACAUUUCUUCUACCAUGUCUCAAUACUCCCGUUCUACGCGAUCAAUGCUUAAACGGCGAGAUUCUACAUAUGGCCCCCUUCAGACACCUGUUGGUUGUGUCCUAGGACUUGUUAAUUCCUCUUUGUUCGUAGAGGACUGCAAGUUGCCUCUCGGUCAAUCUCUCUGUGCUCUUCCUGACACACCUAGUACUCUCACACCUCGCAGUGUGCGCUCUGUGCCAGGUGAAAUGCGCUUGGCAUCCAUUGCUUUUAAUCAUCCCAUUCAGGUUGAUCAACUUAAUAGUAGUUAUUUUAAAUUAAGUAUACCCACUAAUUUUUCCUUUGGUGUGACUCAGGAGUACAUUCAGACAACCAUUCAGAAAGUUACUGUUGAUUGUAAACAGUACGUUUGCAAUGGUUUCCAGAAGUGUGAGCAAUUACUGCGCGAGUAUGGCCAGUUUUGUUCCAAAAUAAACCAGGCUCUCCAUGGUGCCAAUUUACGCCAGGAUGAUUCUGUACGUAAUUUGUUUGCGAGCGUGAAAAGCUCUCAAUCAUCUCCUAUCAUACCAGGUUUUGGAGGUGACUUUAAUUUGACACUUCUAGAACCUGUUUCUAUAUCUACUGGCAGUCGUAGUGCACGUAGUGCUAUUGAGGAUUUGCUAUUUGACAAAGUCACUAUAGCUGAUCCUGGUUAUAUGCAAGGUUACGAUGAUUGUAUGCAGCAAGGUCCAGCAUCAGCUCGUGAUCUUAUUUGUGCUCAAUAUGUGGCUGGUUAUAAAGUAUUACCUCCUCUUAUGGAUGUUAAUAUGGAAGCCGCGUAUACUUCAUCUUUGCUUGGCAGCAUAGCAGGUGUUGGCUGGACUGCUGGCUUAUCCUCCUUUGCUGCUAUUCCAUUUGCACAGAGUAUYUUUUAUAGGUUAAACGGUGUUGGCAUUACUCAACAGGUUCUUUCAGAGAACCAAAAGCUUAUUGCCAAUAAGUUUAAUCAGGCUCUGGGAGCUAUGCAAACAGGCUUCACUACAACUAAUGAAGCUUUUCGGAAGGUUCAGGAUGCUGUGAACAACAAUGCACAGGCUCUAUCCAAAUUAGCUAGCGAGCUAUCUAAUACUUUUGGUGCUAUUUCCGCCUCUAUUGGAGACAUCAUACAACGUCUUGAUGUUCUCGAACAGGACGCCCAAAUAGACAGACUUAUUAAUGGCCGUUUGACAACACUAAAUGCUUUUGUUGCACAGCAGCUUGUUCGUUCCGAAUCAGCUGCUCUUUCCGCUCAAUUGGCUAAAGAUAAAGUCAAUGAGUGUGUCAAGGCACAAUCCAAGCGUUCUGGAUUUUGCGGUCAAGGCACACAUAUAGUGUCCUUUGUUGUAAAUGCCCCUAAUGGCCUUUACUUUAUGCAUGUUGGUUAUUACCCUAGCAACCACAUUGAGGUUGUUUCUGCUUAUGGUCUUUGCGAUGCAGCUAACCCUACUAAUUGUAUAGCCCCUGUUAAUGGCUACUUUAUUAAAACUAAUAACACUAGGAUUGUUGAUGAGUGGUCAUAUACUGGCUCGUCCUUCUAUGCACCUGAGCCCAUCACCUCUCUUAAUACUAAGUAUGUUGCACCACAGGUGACAUACCAAAACAUUUCUACUAACCUCCCUCCUCCUCUUCUCGGCAAUUCCACCGGGAUUGACUUCCAAGAUGAGUUGGAUGAGUUUUUCAAAAAUGUUAGCACCAGUAUACCUAAUUUUGGUUCUCUAACACAGAUUAAUACUACAUUACUCGAUCUUACCUACGAGAUGUUGUCUCUUCAACAAGUUGUUAAAGCCCUUAAUGAGUCUUACAUAGACCUUAAAGAGCUUGGCAAUUAUACUUAUUACAACAAAUGGCCGUGGUACAUUUGGCUUGGUUUCAUUGCUGGGCUUGUUGCCUUAGCUCUAUGCGUCUUCUUCAUACUGUGCUGCACUGGUUGUGGCACAAACUGUAUGGGAAAACUUAAGUGUAAUCGUUGUUGUGAUAGAUACGAGGAAUACGACCUCGAGCCGCAUAAGGUUCAUGU UCACUAA MERS S FLAUGAUACACUCAGUGUUUCUACUGAUGUUCUUGUUAAC 66 SPIKEACCUACAGAAAGUUACGUUGAUGUAGGGCCAGAUUCUG 2cEMC/2012UUAAGUCUGCUUGUAUUGAGGUUGAUAUACAACAGACU (XBaI changeUUCUUUGAUAAAACUUGGCCUAGGCCAAUUGAUGUUUC (U to G)) UAAGGCUGACGGUAUUAUAUACCCUCAAGGCCGUACAU (nucleotide)AUUCUAACAUAACUAUCACUUAUCAAGGUCUUUUUCCCUAUCAGGGAGACCAUGGUGAUAUGUAUGUUUACUCUGCAGGACAUGCUACAGGCACAACUCCACAAAAGUUGUUUGUAGCUAACUAUUCUCAGGACGUCAAACAGUUUGCUAAUGGGUUUGUCGUCCGUAUAGGAGCAGCUGCCAAUUCCACUGGCACUGUUAUUAUUAGCCCAUCUACCAGCGCUACUAUACGAAAAAUUUACCCUGCUUUUAUGCUGGGUUCUUCAGUUGGUAAUUUCUCAGAUGGUAAAAUGGGCCGCUUCUUCAAUCAUACUCUAGUUCUUUUGCCCGAUGGAUGUGGCACUUUACUUAGAGCUUUUUAUUGUAUUCUGGAGCCUCGCUCUGGAAAUCAUUGUCCUGCUGGCAAUUCCUAUACUUCUUUUGCCACUUAUCACACUCCUGCAACAGAUUGUUCUGAUGGCAAUUACAAUCGUAAUGCCAGUCUGAACUCUUUUAAGGAGUAUUUUAAUUUACGUAACUGCACCUUUAUGUACACUUAUAACAUUACCGAAGAUGAGAUUUUAGAGUGGUUUGGCAUUACACAAACUGCUCAAGGUGUUCACCUCUUCUCAUCUCGGUAUGUUGAUUUGUACGGCGGCAAUAUGUUUCAAUUUGCCACCUUGCCUGUUUAUGAUACUAUUAAGUAUUAUUCUAUCAUUCCUCACAGUAUUCGUUCUAUCCAAAGUGAUAGAAAAGCUUGGGCUGCCUUCUACGUAUAUAAACUUCAACCGUUAACUUUCCUGUUGGAUUUUUCUGUUGAUGGUUAUAUACGCAGAGCUAUAGACUGUGGUUUUAAUGAUUUGUCACAACUCCACUGCUCAUAUGAAUCCUUCGAUGUUGAAUCUGGAGUUUAUUCAGUUUCGUCUUUCGAAGCAAAACCUUCUGGCUCAGUUGUGGAACAGGCUGAAGGUGUUGAAUGUGAUUUUUCACCUCUUCUGUCUGGCACACCUCCUCAGGUUUAUAAUUUCAAGCGUUUGGUUUUUACCAAUUGCAAUUAUAAUCUUACCAAAUUGCUUUCACUUUUUUCUGUGAAUGAUUUUACUUGUAGUCAAAUAUCUCCAGCAGCAAUUGCUAGCAACUGUUAUUCUUCACUGAUUUUGGAUUACUUUUCAUACCCACUUAGUAUGAAAUCCGAUCUCAGUGUUAGUUCUGCUGGUCCAAUAUCCCAGUUUAAUUAUAAACAGUCCUUUUCUAAUCCCACAUGUUUGAUUUUAGCGACUGUUCCUCAUAACCUUACUACUAUUACUAAGCCUCUUAAGUACAGCUAUAUUAACAAGUGCUCUCGUCUUCUUUCUGAUGAUCGUACUGAAGUACCUCAGUUAGUGAACGCUAAUCAAUACUCACCCUGUGUAUCCAUUGUCCCAUCCACUGUGUGGGAAGACGGUGAUUAUUAUAGGAAACAACUAUCUCCACUUGAAGGUGGUGGCUGGCUUGUUGCUAGUGGCUCAACUGUUGCCAUGACUGAGCAAUUACAGAUGGGCUUUGGUAUUACAGUUCAAUAUGGUACAGACACCAAUAGUGUUUGCCCCAAGCUUGAAUUUGCUAAUGACACAAAAAUUGCCUCUCAAUUAGGCAAUUGCGUGGAAUAUUCCCUCUAUGGUGUUUCGGGCCGUGGUGUUUUUCAGAAUUGCACAGCUGUAGGUGUUCGACAGCAGCGCUUUGUUUAUGAUGCGUACCAGAAUUUAGUUGGCUAUUAUUCUGAUGAUGGCAACUACUACUGUUUGCGUGCUUGUGUUAGUGUUCCUGUUUCUGUCAUCUAUGAUAAAGAAACUAAAACCCACGCUACUCUAUUUGGUAGUGUUGCAUGUGAACACAUUUCUUCUACCAUGUCUCAAUACUCCCGUUCUACGCGAUCAAUGCUUAAACGGCGAGAUUCUACAUAUGGCCCCCUUCAGACACCUGUUGGUUGUGUCCUAGGACUUGUUAAUUCCUCUUUGUUCGUAGAGGACUGCAAGUUGCCUCUUGGUCAAUCUCUCUGUGCUCUUCCUGACACACCUAGUACUCUCACACCUCGCAGUGUGCGCUCUGUUCCAGGUGAAAUGCGCUUGGCAUCCAUUGCUUUUAAUCAUCCUAUUCAGGUUGAUCAACUUAAUAGUAGUUAUUUUAAAUUAAGUAUACCCACUAAUUUUUCCUUUGGUGUGACUCAGGAGUACAUUCAGACAACCAUUCAGAAAGUUACUGUUGAUUGUAAACAGUACGUUUGCAAUGGUUUCCAGAAGUGUGAGCAAUUACUGCGCGAGUAUGGCCAGUUUUGUUCCAAAAUAAACCAGGCUCUCCAUGGUGCCAAUUUACGCCAGGAUGAUUCUGUACGUAAUUUGUUUGCGAGCGUGAAAAGCUCUCAAUCAUCUCCUAUCAUACCAGGUUUUGGAGGUGACUUUAAUUUGACACUUCUGGAACCUGUUUCUAUAUCUACUGGCAGUCGUAGUGCACGUAGUGCUAUUGAGGAUUUGCUAUUUGACAAAGUCACUAUAGCUGAUCCUGGUUAUAUGCAAGGUUACGAUGAUUGCAUGCAGCAAGGUCCAGCAUCAGCUCGUGAUCUUAUUUGUGCUCAAUAUGUGGCUGGUUACAAAGUAUUACCUCCUCUUAUGGAUGUUAAUAUGGAAGCCGCGUAUACUUCAUCUUUGCUUGGCAGCAUAGCAGGUGUUGGCUGGACUGCUGGCUUAUCCUCCUUUGCUGCUAUUCCAUUUGCACAGAGUAUCUUUUAUAGGUUAAACGGUGUUGGCAUUACUCAACAGGUUCUUUCAGAGAACCAAAAGCUUAUUGCCAAUAAGUUUAAUCAGGCUCUGGGAGCUAUGCAAACAGGCUUCACUACAACUAAUGAAGCUUUUCAGAAGGUUCAGGAUGCUGUGAACAACAAUGCACAGGCUCUAUCCAAAUUAGCUAGCGAGCUAUCUAAUACUUUUGGUGCUAUUUCCGCCUCUAUUGGAGACAUCAUACAACGUCUUGAUGUUCUCGAACAGGACGCCCAAAUAGACAGACUUAUUAAUGGCCGUUUGACAACACUAAAUGCUUUUGUUGCACAGCAGCUUGUUCGUUCCGAAUCAGCUGCUCUUUCCGCUCAAUUGGCUAAAGAUAAAGUCAAUGAGUGUGUCAAGGCACAAUCCAAGCGUUCUGGAUUUUGCGGUCAAGGCACACAUAUAGUGUCCUUUGUUGUAAAUGCCCCUAAUGGCCUUUACUUCAUGCAUGUUGGUUAUUACCCUAGCAACCACAUUGAGGUUGUUUCUGCUUAUGGUCUUUGCGAUGCAGCUAACCCUACUAAUUGUAUAGCCCCUGUUAAUGGCUACUUUAUUAAAACUAAUAACACUAGGAUUGUUGAUGAGUGGUCAUAUACUGGCUCGUCCUUCUAUGCACCUGAGCCCAUUACCUCCCUUAAUACUAAGUAUGUUGCACCACAGGUGACAUACCAAAACAUUUCUACUAACCUCCCUCCUCCUCUUCUCGGCAAUUCCACCGGGAUUGACUUCCAAGAUGAGUUGGAUGAGUUUUUCAAAAAUGUUAGCACCAGUAUACCUAAUUUUGGUUCCCUAACACAGAUUAAUACUACAUUACUCGAUCUUACCUACGAGAUGUUGUCUCUUCAACAAGUUGUUAAAGCCCUUAAUGAGUCUUACAUAGACCUUAAAGAGCUUGGCAAUUAUACUUAUUACAACAAAUGGCCGUGGUACAUUUGGCUUGGUUUCAUUGCUGGGCUUGUUGCCUUAGCUCUAUGCGUCUUCUUCAUACUGUGCUGCACUGGUUGUGGCACAAACUGUAUGGGAAAACUUAAGUGUAAUCGUUGUUGUGAUAGAUACGAGGAAUACGACCUCGAGCCGCAUAAGGUUCAUGUUC ACUAA Novel_MERS_S2_sub-AUGAUCCACUCCGUGUUCCUCCUCAUGUUCCUGUUGACC 67 unit_trimericCCCACUGAGUCAGACUGCAAGCUCCCGCUGGGACAGUCC vaccineCUGUGUGCGCUGCCUGACACUCCUAGCACUCUGACCCCA (nucleotide)CGCUCCGUGCGGUCGGUGCCUGGCGAAAUGCGGCUGGCCUCCAUCGCCUUCAAUCACCCAAUCCAAGUGGAUCAGCUGAAUAGCUCGUAUUUCAAGCUGUCCAUCCCCACGAACUUCUCGUUCGGGGUCACCCAGGAGUACAUCCAGACCACAAUUCAGAAGGUCACCGUCGAUUGCAAGCAAUACGUGUGCAACGGCUUCCAGAAGUGCGAGCAGCUGCUGAGAGAAUACGGGCAGUUUUGCAGCAAGAUCAACCAGGCGCUGCAUGGAGCUAACUUGCGCCAGGACGACUCCGUGCGCAACCUCUUUGCCUCUGUGAAGUCAUCCCAGUCCUCCCCAAUCAUCCCGGGAUUCGGAGGGGACUUCAACCUGACCCUCCUGGAGCCCGUGUCGAUCAGCACCGGUAGCAGAUCGGCGCGCUCAGCCAUUGAAGAUCUUCUGUUCGACAAGGUCACCAUCGCCGAUCCGGGCUACAUGCAGGGAUACGACGACUGUAUGCAGCAGGGACCAGCCUCCGCGAGGGACCUCAUCUGCGCGCAAUACGUGGCCGGGUACAAAGUGCUGCCUCCUCUGAUGGAUGUGAACAUGGAGGCCGCUUAUACUUCGUCCCUGCUCGGCUCUAUCGCCGGCGUGGGGUGGACCGCCGGCCUGUCCUCCUUCGCCGCUAUCCCCUUUGCACAAUCCAUUUUCUACCGGCUCAACGGCGUGGGCAUUACUCAACAAGUCCUGUCGGAGAACCAGAAGUUGAUCGCAAACAAGUUCAAUCAGGCCCUGGGGGCCAUGCAGACUGGAUUCACUACGACUAACGAAGCGUUCCAGAAGGUCCAGGACGCUGUGAACAACAACGCCCAGGCGCUCUCAAAGCUGGCCUCCGAACUCAGCAACACCUUCGGAGCCAUCAGCGCAUCGAUCGGUGACAUAAUUCAGCGGCUGGACGUGCUGGAGCAGGACGCCCAGAUCGACCGCCUCAUCAACGGACGGCUGACCACCUUGAAUGCCUUCGUGGCACAACAGCUGGUCCGGAGCGAAUCAGCGGCACUUUCCGCCCAACUCGCCAAGGACAAAGUCAACGAAUGCGUGAAGGCCCAGUCCAAGAGGUCCGGUUUCUGCGGUCAAGGAACCCAUAUUGUGUCCUUCGUCGUGAACGCGCCCAACGGUCUGUACUUUAUGCACGUCGGCUACUACCCGAGCAAUCAUAUCGAAGUGGUGUCCGCCUACGGCCUGUGCGAUGCCGCUAACCCCACUAACUGUAUUGCCCCUGUGAACGGAUAUUUUAUUAAGACCAACAACACCCGCAUUGUGGACGAAUGGUCAUACACCGGUUCGUCCUUCUACGCGCCCGAGCCCAUCACUUCACUGAACACCAAAUACGUGGCUCCGCAAGUGACCUACCAGAACAUCUCCACCAAUUUGCCGCCGCCGCUGCUCGGAAACAGCACCGGAAUUGAUUUCCAAGAUGAACUGGACGAAUUCUUCAAGAACGUGUCCACUUCCAUUCCCAACUUCGGAAGCCUGACACAGAUCAACACCACCCUUCUCGACCUGACCUACGAGAUGCUGAGCCUUCAACAAGUGGUCAAGGCCCUGAACGAGAGCUACAUCGACCUGAAGGAGCUGGGCAACUAUACCUACUACAACAAGUGGCCGGACAAGAUUGAGGAGAUUCUGUCGAAAAUCUACCACAUUGAAAACGAGAUCGCCAGAAUCA AGAAGCUUAUCGGCGAAGCCMERS_S0_Full- AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUG 68 length SpikeUGGCUGCCUGAUACCACCGGCAGCUAUGUGGACGUGGGC proteinCCCGAUAGCGUGAAGUCCGCCUGUAUCGAAGUGGACAUC (nucleotide,CAGCAGACCUUUUUCGACAAGACCUGGCCCAGACCCAUC codonGACGUGUCCAAGGCCGACGGCAUCAUCUAUCCACAAGGC optimized)CGGACCUACAGCAACAUCACCAUUACCUACCAGGGCCUGUUCCCAUAUCAAGGCGACCACGGCGAUAUGUACGUGUACUCUGCCGGCCACGCCACCGGCACCACACCCCAGAAACUGUUCGUGGCCAACUACAGCCAGGACGUGAAGCAGUUCGCCAACGGCUUCGUCGUGCGGAUUGGCGCCGCUGCCAAUAGCACCGGCACAGUGAUCAUCAGCCCCAGCACCAGCGCCACCAUCCGGAAGAUCUACCCCGCCUUCAUGCUGGGCAGCUCCGUGGGCAAUUUCAGCGACGGCAAGAUGGGCCGGUUCUUCAACCACACCCUGGUGCUGCUGCCCGAUGGCUGUGGCACACUGCUGAGAGCCUUCUACUGCAUCCUGGAACCCAGAAGCGGCAACCACUGCCCUGCCGGCAAUAGCUACACCAGCUUCGCCACCUACCACACACCCGCCACCGAUUGCUCCGACGGCAACUACAACCGGAACGCCAGCCUGAACAGCUUCAAAGAGUACUUCAACCUGCGGAACUGCACCUUCAUGUACACCUACAAUAUCACCGAGGACGAGAUCCUGGAAUGGUUCGGCAUCACCCAGACCGCCCAGGGCGUGCACCUGUUCAGCAGCAGAUACGUGGACCUGUACGGCGGCAACAUGUUCCAGUUUGCCACCCUGCCCGUGUACGACACCAUCAAGUACUACAGCAUCAUCCCCCACAGCAUCCGGUCCAUCCAGAGCGACAGAAAAGCCUGGGCCGCCUUCUACGUGUACAAGCUGCAGCCCCUGACCUUCCUGCUGGACUUCAGCGUGGACGGCUACAUCAGACGGGCCAUCGACUGCGGCUUCAACGACCUGAGCCAGCUGCACUGCUCCUACGAGAGCUUCGACGUGGAAAGCGGCGUGUACAGCGUGUCCAGCUUCGAGGCCAAGCCUAGCGGCAGCGUGGUGGAACAGGCUGAGGGCGUGGAAUGCGACUUCAGCCCUCUGCUGAGCGGCACCCCUCCCCAGGUGUACAACUUCAAGCGGCUGGUGUUCACCAACUGCAAUUACAACCUGACCAAGCUGCUGAGCCUGUUCUCCGUGAACGACUUCACCUGUAGCCAGAUCAGCCCUGCCGCCAUUGCCAGCAACUGCUACAGCAGCCUGAUCCUGGACUACUUCAGCUACCCCCUGAGCAUGAAGUCCGAUCUGAGCGUGUCCUCCGCCGGACCCAUCAGCCAGUUCAACUACAAGCAGAGCUUCAGCAACCCUACCUGCCUGAUUCUGGCCACCGUGCCCCACAAUCUGACCACCAUCACCAAGCCCCUGAAGUACAGCUACAUCAACAAGUGCAGCAGACUGCUGUCCGACGACCGGACCGAAGUGCCCCAGCUCGUGAACGCCAACCAGUACAGCCCCUGCGUGUCCAUCGUGCCCAGCACCGUGUGGGAGGACGGCGACUACUACAGAAAGCAGCUGAGCCCCCUGGAAGGCGGCGGAUGGCUGGUGGCUUCUGGAAGCACAGUGGCCAUGACCGAGCAGCUGCAGAUGGGCUUUGGCAUCACCGUGCAGUACGGCACCGACACCAACAGCGUGUGCCCCAAGCUGGAAUUCGCCAAUGACACCAAGAUCGCCAGCCAGCUGGGAAACUGCGUGGAAUACUCCCUGUAUGGCGUGUCCGGACGGGGCGUGUUCCAGAAUUGCACAGCAGUGGGAGUGCGGCAGCAGAGAUUCGUGUACGAUGCCUACCAGAACCUCGUGGGCUACUACAGCGACGACGGCAAUUACUACUGCCUGCGGGCCUGUGUGUCCGUGCCCGUGUCCGUGAUCUACGACAAAGAGACAAAGACCCACGCCACACUGUUCGGCUCCGUGGCCUGCGAGCACAUCAGCUCCACCAUGAGCCAGUACUCCCGCUCCACCCGGUCCAUGCUGAAGCGGAGAGAUAGCACCUACGGCCCCCUGCAGACACCUGUGGGAUGUGUGCUGGGCCUCGUGAACAGCUCCCUGUUUGUGGAAGAUUGCAAGCUGCCCCUGGGCCAGAGCCUGUGUGCCCUGCCAGAUACCCCUAGCACCCUGACCCCUAGAAGCGUGCGCUCUGUGCCCGGCGAAAUGCGGCUGGCCUCUAUCGCCUUCAAUCACCCCAUCCAGGUGGACCAGCUGAACUCCAGCUACUUCAAGCUGAGCAUCCCCACCAACUUCAGCUUCGGCGUGACCCAGGAGUACAUCCAGACCACAAUCCAGAAAGUGACCGUGGACUGCAAGCAGUACGUGUGCAACGGCUUUCAGAAGUGCGAACAGCUGCUGCGCGAGUACGGCCAGUUCUGCAGCAAGAUCAACCAGGCCCUGCACGGCGCCAACCUGAGACAGGAUGACAGCGUGCGGAACCUGUUCGCCAGCGUGAAAAGCAGCCAGUCCAGCCCCAUCAUCCCUGGCUUCGGCGGCGACUUUAACCUGACCCUGCUGGAACCUGUGUCCAUCAGCACCGGCUCCAGAAGCGCCAGAUCCGCCAUCGAGGACCUGCUGUUCGACAAAGUGACCAUUGCCGACCCCGGCUACAUGCAGGGCUACGACGAUUGCAUGCAGCAGGGCCCAGCCAGCGCCAGGGAUCUGAUCUGUGCCCAGUAUGUGGCCGGCUACAAGGUGCUGCCCCCCCUGAUGGACGUGAACAUGGAAGCCGCCUACACCUCCAGCCUGCUGGGCUCUAUUGCUGGCGUGGGAUGGACAGCCGGCCUGUCUAGCUUUGCCGCCAUCCCUUUCGCCCAGAGCAUCUUCUACCGGCUGAACGGCGUGGGCAUCACACAACAGGUGCUGAGCGAGAACCAGAAGCUGAUCGCCAACAAGUUUAACCAGGCACUGGGCGCCAUGCAGACCGGCUUCACCACCACCAACGAGGCCUUCAGAAAGGUGCAGGACGCCGUGAACAACAACGCCCAGGCUCUGAGCAAGCUGGCCUCCGAGCUGAGCAAUACCUUCGGCGCCAUCAGCGCCUCCAUCGGCGACAUCAUCCAGCGGCUGGACGUGCUGGAACAGGACGCCCAGAUCGACCGGCUGAUCAACGGCAGACUGACCACCCUGAACGCCUUCGUGGCACAGCAGCUCGUGCGGAGCGAAUCUGCCGCUCUGUCUGCUCAGCUGGCCAAGGACAAAGUGAACGAGUGCGUGAAGGCCCAGUCCAAGCGGAGCGGCUUUUGUGGCCAGGGCACCCACAUCGUGUCCUUCGUCGUGAAUGCCCCCAACGGCCUGUACUUUAUGCACGUGGGCUAUUACCCCAGCAACCACAUCGAGGUGGUGUCCGCCUAUGGCCUGUGCGACGCCGCCAAUCCUACCAACUGUAUCGCCCCCGUGAACGGCUACUUCAUCAAGACCAACAACACCCGGAUCGUGGACGAGUGGUCCUACACAGGCAGCAGCUUCUACGCCCCCGAGCCCAUCACCUCCCUGAACACCAAAUACGUGGCCCCCCAAGUGACAUACCAGAACAUCUCCACCAACCUGCCCCCUCCACUGCUGGGAAAUUCCACCGGCAUCGACUUCCAGGACGAGCUGGACGAGUUCUUCAAGAACGUGUCCACCUCCAUCCCCAACUUCGGCAGCCUGACCCAGAUCAACACCACUCUGCUGGACCUGACCUACGAGAUGCUGUCCCUGCAACAGGUCGUGAAAGCCCUGAACGAGAGCUACAUCGACCUGAAAGAGCUGGGGAACUACACCUACUACAACAAGUGGCCUUGGUACAUUUGGCUGGGCUUUAUCGCCGGCCUGGUGGCCCUGGCCCUGUGCGUGUUCUUCAUCCUGUGCUGCACCGGCUGCGGCACCAAUUGCAUGGGCAAGCUGAAAUGCAACCGGUGCUGCGACAGAUACGAGGAAUACGACCUGG AACCUCACAAAGUGCAUGUGCAC

TABLE 11 Betacoronavirus Amino Acid Sequences SEQ ID StrainAmino Acid Sequence NO: gb|KJ156934.1|:MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDK 24 21405-25466 TWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDM Middle EastYVYSAGHATGTTpQKLFVANYSQDVKQFANGFVVRIGAAANS respiratoryTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHT syndromeLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTP coronavirusATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILE isolateWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYS Riyadh_14_2013,IIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRA spike proteinIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQA (amino acid)EGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFtCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINqALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFrKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYD LEPHKVHVH MERS S FLMIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDK 25 SPIKETWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDM 2cEMC/2012YVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANS (XBaI changeTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHT (T to G)) LVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTP (amino acid)ATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYD LEPHKVHVH Novel_MERS_S2_sub-MIHSVFLLMFLLTPTESDCKLPLGQSLCALPDTPSTLTPRSV 26 unit_trimericRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQ vaccine (aminoEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQA acid)LHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWP DKIEEILSKIYHIENEIARIKKLIGEAIsolate A1- MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDK 27 Hasa_1_2013TWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDM (NCBI accessionYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANS #AGN70962)TGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPHVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYD LEPHKVHVH Middle EastMIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDK 28 respiratoryTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDM syndromeYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANS coronavirus STGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHT proteinLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTP UniProtKB-ATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNITEDEILE R9UQ53WFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPHVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYD LEPHKVHVH Human SARSMFIFLLFLTLTSGSDLDRCTTFDDVQAPNYTQHTSSMRGVYY 29 coronavirusPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFGNPVIPFK (SARS-CoV)DGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIR (Severe acuteACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYISDAF respiratorySLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLP syndromeSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAY coronavirus)FVGYLKPTTFMLKYDENGTITDAVDCSQNPLAELKCSVKSFE SpikeIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPS glycoproteinVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLC UniProtKB-FSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCV P59594LAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMSLGADSSIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMYICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYKTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT HumanMFLILLISLPTAFAVIGDLKCTSDNINDKDTGPPPISTDTVD 30 coronavirus OC43VTNGLGTYYVLDRVYLNTTLFLNGYYPTSGSTYRNMALKGSV (HCoV-OC43)LLSRLWFKPPFLSDFINGIFAKVKNTKVIKDRVMYSEFPAIT SpikeIGSTFVNTSYSVVVQPRTINSTQDGDNKLQGLLEVSVCQYNM glycoproteinCEYPQTICHPNLGNHRKELWHLDTGVVSCLYKRNFTYDVNAD UniProtKB-YLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGMALSHYYVM P36334PLTCNSKLTLEYWVTPLTSRQYLLAFNQDGIIFNAEDCMSDFMSEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPNLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPRPAGVLTNHDVVYAQHCFKAPKNFCPCKLNGSCVGSGPGKNNGIGTCPAGTNYLTCDNLCTPDPITFTGTYKCPQTKSLVGIGEHCSGLAVKSDYCGGNSCTCRPQAFLGWSADSCLQGDKCNIFANFILHDVNSGLTCSTDLQKANTDIILGVCVNYDLYGILGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYIINRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNSLTRQLQPINYFDSYLGCVVNAYNSTAISVQTCDLTVGSGYCVDYSKNRRSRGAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIPSEFTIGNMVEFIQTSSPKVTIDCAAFVCGDYAACKSQLVEYGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSPVLGCLGSECSKASSRSAIEDLLFDKVKLSDVGFVEAYNNCTGGAEIRDLICVQSYKGIKVLPPLLSENQISGYTLAATSASLFPPWTAAAGVPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALYAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFGAISASLQEILSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTARVSPGLCIAGDRGIAPKSGYFVNVNNTWMYTGSGYYYPEPITENNVVVMSTCAVNYTKAPYVMLNTSIPNLPDFKEELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEAIKVLNQSYINLKDIGTYEYYVKWPWYVWLLICLAGVAMLVLLFFICCCTGCGTSCFKKCGGCCDDYTGYQE LVIKTSHDD HumanMFLIIFILPTTLAVIGDFNCTNSFINDYNKTIPRISEDVVDV 31 coronavirusSLGLGTYYVLNRVYLNTTLLFTGYFPKSGANFRDLALKGSIY HKU1 (isolateLSTLWYKPPFLSDFNNGIFSKVKNTKLYVNNTLYSEFSTIVI N5) (HCoV-GSVFVNTSYTIVVQPHNGILEITACQYTMCEYPHTVCKSKGS HKU1) SpikeIRNESWHIDSSEPLCLFKKNFTYNVSADWLYFHFYQERGVFY glycoproteinAYYADVGMPTTFLFSLYLGTILSHYYVMPLTCNAISSNTDNE UniProtKB-TLEYWVTPLSRRQYLLNFDEHGVITNAVDCSSSFLSEIQCKT Q0ZME7QSFAPNTGVYDLSGFTVKPVATVYRRIPNLPDCDIDNWLNNVSVPSPLNWERRIFSNCNFNLSTLLRLVHVDSFSCNNLDKSKIFGSCFNSITVDKFAIPNRRRDDLQLGSSGFLQSSNYKIDISSSSCQLYYSLPLVNVTINNFNPSSWNRRYGFGSFNLSSYDVVYSDHCFSVNSDFCPCADPSVVNSCAKSKPPSAICPAGTKYRHCDLDTTLYVKNWCRCSCLPDPISTYSPNTCPQKKVVVGIGEHCPGLGINEEKCGTQLNHSSCFCSPDAFLGWSFDSCISNNRCNIFSNFIFNGINSGTTCSNDLLYSNTEISTGVCVNYDLYGITGQGIFKEVSAAYYNNWQNLLYDSNGNIIGFKDFLTNKTYTILPCYSGRVSAAFYQNSSSPALLYRNLKCSYVLNNISFISQPFYFDSYLGCVLNAVNLTSYSVSSCDLRMGSGFCIDYALPSSRRKRRGISSPYRFVTFEPFNVSFVNDSVETVGGLFEIQIPTNFTIAGHEEFIQTSSPKVTIDCSAFVCSNYAACHDLLSEYGTFCDNINSILNEVNDLLDITQLQVANALMQGVTLSSNLNTNLHSDVDNIDFKSLLGCLGSQCGSSSRSLLEDLLFNKVKLSDVGFVEAYNNCTGGSEIRDLLCVQSFNGIKVLPPILSETQISGYTTAATVAAMFPPWSAAAGVPFSLNVQYRINGLGVTMDVLNKNQKLIANAFNKALLSIQNGFTATNSALAKIQSVVNANAQALNSLLQQLFNKFGAISSSLQEILSRLDNLEAQVQIDRLINGRLTALNAYVSQQLSDITLIKAGASRAIEKVNECVKSQSPRINFCGNGNHILSLVQNAPYGLLFIHFSYKPTSFKTVLVSPGLCLSGDRGIAPKQGYFIKQNDSWMFTGSSYYYPEPISDKNVVFMNSCSVNFTKAPFIYLNNSIPNLSDFEAELSLWFKNHTSIAPNLTFNSHINATFLDLYYEMNVIQESIKSLNSSFINLKEIGTYEMYVKWPWYIWLLIVILFIIFLMILFFICCCTGCGSACFSKCHNCCDEYGGHNDFV IKASHDD Novel_SARS_S2MFIFLLFLTLTSGSDLDRALSGIAAEQDRNTREVFAQVKQMY 32KTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCG SCCKFDEDDSEPVLKGVKLHYTNovel_MERS_S2 MIHSVFLLMFLLTPTESDCKLPLGQSLCALPDTPSTLTPRSV 33RSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTIQKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQSSPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASARDLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNGVGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNECVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCIAPVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTGIDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYTYYNKWP Novel_Trimeric_SARS_S2MFIFLLFLTLTSGSDLDRALSGIAAEQDRNTREVFAQVKQMY 34KTPTLKYFGGFNFSQILPDPLKPTKRSFIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLLTDDMIAAYTAALVSGTATAGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQIITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCG SCCKFDEDDSEPVLKGVKLHYT

TABLE 12 Full-length Spike Glycoprotein Amino Acid Sequences (Homosapiens strains) GenBank Accession Country Collection Date Release DateVirus Name AFY13307 United 2012 Sep. 11 2012 Dec. 5 BetacoronavirusEngland 1, Kingdom complete genome AFS88936 2012 Jun. 13 2012 Sep. 27Human betacoronavirus 2c EMC/2012, complete genome AGG22542 United 2012Sep. 19 2013 Feb. 27 Human betacoronavirus 2c England- KingdomQatar/2012, complete genome AHY21469 Jordan 2012 2014 May 4 Humanbetacoronavirus 2c Jordan- N3/2012 isolate MG167, complete genomeAGH58717 Jordan 2012 April 2013 Mar. 25 Human betacoronavirus 2c Jordan-N3/2012, complete genome AGV08444 Saudi 2013 May 7 2013 Sep. 17 MiddleEast respiratory syndrome Arabia coronavirus isolate Al- Hasa_12_2013,complete genome AGV08546 Saudi 2013 May 11 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_15_2013,complete genome AGV08535 Saudi 2013 May 12 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_16_2013,complete genome AGV08558 Saudi 2013 May 15 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_17_2013,complete genome AGV08573 Saudi 2013 May 23 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_18_2013,complete genome AGV08480 Saudi 2013 May 23 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_19_2013,complete genome AGN70962 Saudi 2013 May 9 2013 Jun. 10 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_1_2013,complete genome AGV08492 Saudi 2013 May 30 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_21_2013,complete genome AHI48517 Saudi 2013 May 2 2014 Feb. 6 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_25_2013,complete genome AGN70951 Saudi 2013 Apr. 21 2013 Jun. 10 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_2_2013,complete genome AGN70973 Saudi 2013 Apr. 22 2013 Jun. 10 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_3_2013,complete genome AGN70929 Saudi 2013 May 1 2013 Jun. 10 Middle Eastrespiratory syndrome Arabia coronavirus isolate Al- Hasa_4_2013,complete genome AGV08408 Saudi 2012 Jun. 19 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Bisha_1_2012, completegenome AGV08467 Saudi 2013 May 13 2013 Sep. 17 Middle East respiratorysyndrome Arabia coronavirus isolate Buraidah_1_2013, complete genomeAID50418 United 2013 Feb. 10 2014 Jun. 18 Middle East respiratorysyndrome Kingdom coronavirus isolate England/2/2013, complete genomeAJD81451 United 2013 Feb. 10 2015 Jan. 18 Middle East respiratorysyndrome Kingdom coronavirus isolate England/3/2013, complete genomeAJD81440 United 2013 Feb. 13 2015 Jan. 18 Middle East respiratorysyndrome Kingdom coronavirus isolate England/4/2013, complete genomeAHB33326 France 2013 May 7 2013 Dec. 7 Middle East respiratory syndromecoronavirus isolate FRA/UAE, complete genome AIZ48760 USA 2014 June 2014Dec. 14 Middle East respiratory syndrome coronavirus isolateFlorida/USA- 2_Saudi Arabia_2014, complete genome AGV08455 Saudi 2013Jun. 4 2013 Sep. 17 Middle East respiratory syndrome Arabia coronavirusisolate Hafr-Al- Batin_1_2013, complete genome AHI48561 Saudi 2013 Aug.5 2014 Feb. 6 Middle East respiratory syndrome Arabia coronavirusisolate Hafr-Al- Batin_2_2013, complete genome AHI48539 Saudi 2013 Aug.28 2014 Feb. 6 Middle East respiratory syndrome Arabia coronavirusisolate Hafr-Al- Batin_6_2013, complete genome AIZ74417 France 2013 Apr.26 2015 Mar. 10 Middle East respiratory syndrome coronavirus isolateHu-France (UAE) - FRA1_1627- 2013_BAL_Sanger, complete genome AIZ74433France 2013 May 7 2015 Mar. 10 Middle East respiratory syndromecoronavirus isolate Hu-France - FRA2_130569-2013_IS_HTS, complete genomeAIZ74439 France 2013 May 7 2015 Mar. 10 Middle East respiratory syndromecoronavirus isolate Hu-France - FRA2_130569-2013_InSpu_Sanger, completegenome AIZ74450 France 2013 May 7 2015 Mar. 10 Middle East respiratorysyndrome coronavirus isolate Hu-France -FRA2_130569-2013_Isolate_Sanger, complete genome AKK52602 Saudi 2015Feb. 10 2015 Jun. 8 Middle East respiratory syndrome Arabia coronavirusisolate Hu/Riyadh_KSA_2959_2015, complete genome AKK52612 Saudi 2015Mar. 1 2015 Jun. 8 Middle East respiratory syndrome Arabia coronavirusisolate Hu/Riyadh_KSA_4050_2015, complete genome AHN10812 Saudi 2013Nov. 6 2014 Mar. 24 Middle East respiratory syndrome Arabia coronavirusisolate Jeddah_1_2013, complete genome AID55071 Saudi 2014 Apr. 21 2014Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C10306/KSA/2014-04-20, complete genome AID55066 Saudi 2014 2014Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C7149/KSA/2014-04-05, complete genome AID55067 Saudi 2014 2014Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C7569/KSA/2014-04-03, complete genome AID55068 Saudi 2014 Apr. 72014 Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C7770/KSA/2014-04-07, complete genome AID55069 Saudi 2014 Apr. 122014 Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C8826/KSA/2014-04-12, complete genome AID55070 Saudi 2014 Apr. 142014 Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateJeddah_C9055/KSA/2014-04-14, complete genome AHE78108 Saudi 2013 Nov. 52014 May 1 Middle East respiratory syndrome Arabia coronavirus isolateMERS-CoV- Jeddah-human-1, complete genome AKL59401 South 2015 May 202015 Jun. 9 Middle East respiratory syndrome Korea coronavirus isolateMERS- CoV/KOR/KNIH/002_05_2015, complete genome ALD51904 Thailand 2015Jun. 17 2015 Jul. 7 Middle East respiratory syndrome coronavirus isolateMERS- CoV/THA/CU/17_06_2015, complete genome AID55072 Saudi 2014 Apr. 152014 Nov. 12 Middle East respiratory syndrome Arabia coronavirus isolateMakkah_C9355/KSA/Makkah/2014- 04-15, complete genome AHC74088 Qatar 2013Oct. 13 2013 Dec. 23 Middle East respiratory syndrome coronavirusisolate Qatar3, complete genome AHC74098 Qatar 2013 Oct. 17 2013 Dec. 23Middle East respiratory syndrome coronavirus isolate Qatar4, completegenome AHI48572 Saudi 2013 Aug. 15 2014 Feb. 6 Middle East respiratorysyndrome Arabia coronavirus isolate Riyadh_14_2013, complete genomeAGV08379 Saudi 2012 Oct. 23 2013 Sep. 17 Middle East respiratorysyndrome Arabia coronavirus isolate Riyadh_1_2012, complete genomeAID55073 Saudi 2014 Apr. 22 2014 Nov. 12 Middle East respiratorysyndrome Arabia coronavirus isolate Riyadh_2014KSA_683/KSA/2014,complete genome AGV08584 Saudi 2012 Oct. 30 2013 Sep. 17 Middle Eastrespiratory syndrome Arabia coronavirus isolate Riyadh_2_2012, completegenome AGV08390 Saudi 2013 Feb. 5 2013 Sep. 17 Middle East respiratorysyndrome Arabia coronavirus isolate Riyadh_3_2013, complete genomeAHI48605 Saudi 2013 Mar. 1 2014 Feb. 6 Middle East respiratory syndromeArabia coronavirus isolate Riyadh_4_2013, complete genome AHI48583 Saudi2013 Jul. 2 2014 Feb. 6 Middle East respiratory syndrome Arabiacoronavirus isolate Riyadh_5_2013, complete genome AHI48528 Saudi 2013Jul. 17 2014 Feb. 6 Middle East respiratory syndrome Arabia coronavirusisolate Riyadh_9_2013, complete genome AHI48594 Saudi 2013 Jun. 12 2014Feb. 6 Middle East respiratory syndrome Arabia coronavirus isolateTaif_1_2013, complete genome AHI48550 Saudi 2013 Jun. 12 2014 Feb. 6Middle East respiratory syndrome Arabia coronavirus isolate Wadi-Ad-Dawasir_1_2013, complete genome AIY60558 United 2014 Mar. 7 2014 Dec. 6Middle East respiratory syndrome Arab coronavirus strain Abu EmiratesDhabi/Gayathi_UAE_2_2014, complete genome AIY60538 United 2014 Apr. 102014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_16_2014, complete genome AIY60528 United 2014 Apr. 102014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_18_2014, complete genome AIY60588 United 2014 Apr. 132014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_26_2014, complete genome AIY60548 United 2014 Apr. 192014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_30_2014, complete genome AIY60568 United 2014 Apr. 172014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_33_2014, complete genome AIY60518 United 2014 Apr. 72014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_8_2014, complete genome AIY60578 United 2013 Nov. 152014 Dec. 6 Middle East respiratory syndrome Arab coronavirus strain AbuEmirates Dhabi_UAE_9_2013, complete genome AKJ80137 China 2015 May 272015 Jun. 5 Middle East respiratory syndrome coronavirus strainChinaGD01, complete genome AHZ64057 USA 2014 May 10 2014 May 14 MiddleEast respiratory syndrome coronavirus strain Florida/USA- 2_SaudiArabia_2014, complete genome AKM76229 Oman 2013 Oct. 28 2015 Jun. 23Middle East respiratory syndrome coronavirus strain Hu/Oman_2285_2013,complete genome AKM76239 Oman 2013 Dec. 28 2015 Jun. 23 Middle Eastrespiratory syndrome coronavirus strain Hu/Oman_2874_2013, completegenome AKI29284 Saudi 2015 Jan. 6 2015 May 27 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh-KSA- 2049/2015, completegenome AKI29265 Saudi 2015 Jan. 21 2015 May 27 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh-KSA- 2343/2015, completegenome AKI29255 Saudi 2015 Jan. 21 2015 May 27 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh-KSA- 2345/2015, completegenome AKI29275 Saudi 2015 Jan. 26 2015 May 27 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh-KSA- 2466/2015, completegenome AKK52582 Saudi 2015 Feb. 10 2015 Jun. 8 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh_KSA_2959_2015, completegenome AKK52592 Saudi 2015 Mar. 1 2015 Jun. 8 Middle East respiratorysyndrome Arabia coronavirus strain Hu/Riyadh_KSA_4050_2015, completegenome AHZ58501 USA 2014 Apr. 30 2014 May 13 Middle East respiratorysyndrome coronavirus strain Indiana/USA- 1_Saudi Arabia_2014, completegenome AGN52936 United 2013 2013 Jun. 10 Middle East respiratorysyndrome Arab coronavirus, complete genome Emirates

TABLE 13 SEQ ID Description Sequence NO: MeV Nucleic Acid SequencesGC_F_MEASLES_B3.1 TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACT 35Sequence, NT (5′ CACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAA UTR, ORF, 3′GAAATATAAGAGCCACCATGGGTCTCAAGGTGAACGTC UTR)TCTGCCGTATTCATGGCAGTACTGTTAACTCTCCAAACA Sequence Length:CCCGCCGGTCAAATTCATTGGGGCAATCTCTCTAAGAT 1864AGGGGTAGTAGGAATAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAATTAATGCCCAATATAACTCTCCTCAATAACTGCACGAGGGTAGAGATTGCAGAATACAGGAGACTACTAAGAACAGTTTTGGAACCAATTAGGGATGCACTTAATGCAATGACCCAGAACATAAGGCCGGTTCAGAGCGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTAGTCCTGGCAGGTGCGGCCCTAGGTGTTGCCACAGCTGCTCAGATAACAGCCGGCATTGCACTTCACCGGTCCATGCTGAACTCTCAGGCCATCGACAATCTGAGAGCGAGCCTGGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAACCAGCTATCTTGTGATCTAATCGGTCAGAAGCTCGGGCTCAAATTGCTTAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCCTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGTTATGCACTTGGAGGAGATATCAATAAGGTGTTAGAAAAGCTCGGATACAGTGGAGGCGATTTACTAGGCATCTTAGAGAGCAGAGGAATAAAGGCTCGGATAACTCACGTCGACACAGAGTCCTACTTCATAGTCCTCAGTATAGCCTATCCGACGCTGTCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGTACTTTCATGCCAGAGGGGACTGTGTGCAGCCAAAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACCAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTTGGGAACCGGTTCATTTTATCACAAGGGAACCTAATAGCCAATTGTGCATCAATTCTTTGTAAGTGTTACACAACAGGTACGATTATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCGCTGCCCGGTAGTCGAGGTGAACGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCAGACGCTGTGTACTTGCACAGAATTGACCTCGGTCCTCCCATATCATTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCCAAATTGGAGGATGCCAAGGAATTGTTGGAATCATCGGACCAGATATTGAGAAGTATGAAAGGTTTATCGAGCACTAGCATAGTCTACATCCTGATTGCAGTGTGTCTTGGAGGGTTGATAGGGATCCCCACTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAAAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGACCTTACAGGAACATCAAAATCCTATGTAAGATCGCTTTGATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC GC_F_MEASLES_B3.1ATGGGTCTCAAGGTGAACGTCTCTGCCGTATTCATGGC 36 ORF Sequence, NTAGTACTGTTAACTCTCCAAACACCCGCCGGTCAAATTCATTGGGGCAATCTCTCTAAGATAGGGGTAGTAGGAATAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAATTAATGCCCAATATAACTCTCCTCAATAACTGCACGAGGGTAGAGATTGCAGAATACAGGAGACTACTAAGAACAGTTTTGGAACCAATTAGGGATGCACTTAATGCAATGACCCAGAACATAAGGCCGGTTCAGAGCGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTAGTCCTGGCAGGTGCGGCCCTAGGTGTTGCCACAGCTGCTCAGATAACAGCCGGCATTGCACTTCACCGGTCCATGCTGAACTCTCAGGCCATCGACAATCTGAGAGCGAGCCTGGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAACCAGCTATCTTGTGATCTAATCGGTCAGAAGCTCGGGCTCAAATTGCTTAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCCTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGTTATGCACTTGGAGGAGATATCAATAAGGTGTTAGAAAAGCTCGGATACAGTGGAGGCGATTTACTAGGCATCTTAGAGAGCAGAGGAATAAAGGCTCGGATAACTCACGTCGACACAGAGTCCTACTTCATAGTCCTCAGTATAGCCTATCCGACGCTGTCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGTACTTTCATGCCAGAGGGGACTGTGTGCAGCCAAAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACCAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTTGGGAACCGGTTCATTTTATCACAAGGGAACCTAATAGCCAATTGTGCATCAATTCTTTGTAAGTGTTACACAACAGGTACGATTATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCGCTGCCCGGTAGTCGAGGTGAACGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCAGACGCTGTGTACTTGCACAGAATTGACCTCGGTCCTCCCATATCATTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCCAAATTGGAGGATGCCAAGGAATTGTTGGAATCATCGGACCAGATATTGAGAAGTATGAAAGGTTTATCGAGCACTAGCATAGTCTACATCCTGATTGCAGTGTGTCTTGGAGGGTTGATAGGGATCCCCACTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAAAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGACCTTACAGGAACATCAAAATCC TATGTAAGATCGCTTTGAGC_F_MEASLES_B3.1 G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAT 37 mRNA SequenceATAAGAGCCACCATGGGTCTCAAGGTGAACGTCTCTGC (assumes T100 tail)CGTATTCATGGCAGTACTGTTAACTCTCCAAACACCCG mRNA SequenceCCGGTCAAATTCATTGGGGCAATCTCTCTAAGATAGGG Length: 1925GTAGTAGGAATAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAATTAATGCCCAATATAACTCTCCTCAATAACTGCACGAGGGTAGAGATTGCAGAATACAGGAGACTACTAAGAACAGTTTTGGAACCAATTAGGGATGCACTTAATGCAATGACCCAGAACATAAGGCCGGTTCAGAGCGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTAGTCCTGGCAGGTGCGGCCCTAGGTGTTGCCACAGCTGCTCAGATAACAGCCGGCATTGCACTTCACCGGTCCATGCTGAACTCTCAGGCCATCGACAATCTGAGAGCGAGCCTGGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAACCAGCTATCTTGTGATCTAATCGGTCAGAAGCTCGGGCTCAAATTGCTTAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCCTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGTTATGCACTTGGAGGAGATATCAATAAGGTGTTAGAAAAGCTCGGATACAGTGGAGGCGATTTACTAGGCATCTTAGAGAGCAGAGGAATAAAGGCTCGGATAACTCACGTCGACACAGAGTCCTACTTCATAGTCCTCAGTATAGCCTATCCGACGCTGTCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGTACTTTCATGCCAGAGGGGACTGTGTGCAGCCAAAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACCAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTTGGGAACCGGTTCATTTTATCACAAGGGAACCTAATAGCCAATTGTGCATCAATTCTTTGTAAGTGTTACACAACAGGTACGATTATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCGCTGCCCGGTAGTCGAGGTGAACGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCAGACGCTGTGTACTTGCACAGAATTGACCTCGGTCCTCCCATATCATTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCCAAATTGGAGGATGCCAAGGAATTGTTGGAATCATCGGACCAGATATTGAGAAGTATGAAAGGTTTATCGAGCACTAGCATAGTCTACATCCTGATTGCAGTGTGTCTTGGAGGGTTGATAGGGATCCCCACTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAAAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGACCTTACAGGAACATCAAAATCCTATGTAAGATCGCTTTGATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAATCTAGGC_F_MEASLES_D8 TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACT 38Sequence, NT (5′ CACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAA UTR, ORF, 3′GAAATATAAGAGCCACCATGGGTCTCAAGGTGAACGTC UTR)TCTGTCATATTCATGGCAGTACTGTTAACTCTTCAAACA Sequence Length:CCCACCGGTCAAATCCATTGGGGCAATCTCTCTAAGAT 1864AGGGGTGGTAGGGGTAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAGTTAATGCCCAATATAACTCTCCTCAACAATTGCACGAGGGTAGGGATTGCAGAATACAGGAGACTACTGAGAACAGTTCTGGAACCAATTAGAGATGCACTTAATGCAATGACCCAGAATATAAGACCGGTTCAGAGTGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTTGTCCTGGCAGGTGCGGCCCTAGGCGTTGCCACAGCTGCTCAAATAACAGCCGGTATTGCACTTCACCAGTCCATGCTGAACTCTCAAGCCATCGACAATCTGAGAGCGAGCCTAGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAATCAACTATCTTGTGATTTAATCGGCCAGAAGCTAGGGCTCAAATTGCTCAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCTTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGCTATGCGCTTGGAGGAGATATCAATAAGGTGTTGGAAAAGCTCGGATACAGTGGAGGTGATCTACTGGGCATCTTAGAGAGCAGAGGAATAAAGGCCCGGATAACTCACGTCGACACAGAGTCCTACTTCATTGTACTCAGTATAGCCTATCCGACGCTATCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGCACTTTCATGCCAGAGGGGACTGTGTGCAGCCAGAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACTAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTCGGGAACCGGTTCATTTTATCACAGGGGAACCTAATAGCCAATTGTGCATCAATCCTTTGCAAGTGTTACACAACAGGAACAATCATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCACTGCCCGGTGGTCGAGGTGAATGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCGGACGCTGTGTACTTGCACAGGATTGACCTCGGTCCTCCCATATCTTTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCTAAGTTGGAGGATGCCAAGGAATTGTTGGAGTCATCGGACCAGATATTGAGGAGTATGAAAGGTTTATCGAGCACTAGTATAGTTTACATCCTGATTGCAGTGTGTCTTGGAGGATTGATAGGGATCCCCGCTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAGAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGATCTTACAGGAACATCAAAATCCTATGTAAGGTCACTCTGATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC GC_F_MEASLES_D8ATGGGTCTCAAGGTGAACGTCTCTGTCATATTCATGGC 39 ORF Sequence, NTAGTACTGTTAACTCTTCAAACACCCACCGGTCAAATCCATTGGGGCAATCTCTCTAAGATAGGGGTGGTAGGGGTAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAGTTAATGCCCAATATAACTCTCCTCAACAATTGCACGAGGGTAGGGATTGCAGAATACAGGAGACTACTGAGAACAGTTCTGGAACCAATTAGAGATGCACTTAATGCAATGACCCAGAATATAAGACCGGTTCAGAGTGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTTGTCCTGGCAGGTGCGGCCCTAGGCGTTGCCACAGCTGCTCAAATAACAGCCGGTATTGCACTTCACCAGTCCATGCTGAACTCTCAAGCCATCGACAATCTGAGAGCGAGCCTAGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAATCAACTATCTTGTGATTTAATCGGCCAGAAGCTAGGGCTCAAATTGCTCAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCTTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGCTATGCGCTTGGAGGAGATATCAATAAGGTGTTGGAAAAGCTCGGATACAGTGGAGGTGATCTACTGGGCATCTTAGAGAGCAGAGGAATAAAGGCCCGGATAACTCACGTCGACACAGAGTCCTACTTCATTGTACTCAGTATAGCCTATCCGACGCTATCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGCACTTTCATGCCAGAGGGGACTGTGTGCAGCCAGAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACTAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTCGGGAACCGGTTCATTTTATCACAGGGGAACCTAATAGCCAATTGTGCATCAATCCTTTGCAAGTGTTACACAACAGGAACAATCATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCACTGCCCGGTGGTCGAGGTGAATGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCGGACGCTGTGTACTTGCACAGGATTGACCTCGGTCCTCCCATATCTTTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCTAAGTTGGAGGATGCCAAGGAATTGTTGGAGTCATCGGACCAGATATTGAGGAGTATGAAAGGTTTATCGAGCACTAGTATAGTTTACATCCTGATTGCAGTGTGTCTTGGAGGATTGATAGGGATCCCCGCTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAGAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGATCTTACAGGAACATCAAAATCCT ATGTAAGGTCACTCTGA GC_F_MEASLES_D8G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAT 40 mRNA SequenceATAAGAGCCACCATGGGTCTCAAGGTGAACGTCTCTGT (assumes T100 tail)CATATTCATGGCAGTACTGTTAACTCTTCAAACACCCAC Sequence Length:CGGTCAAATCCATTGGGGCAATCTCTCTAAGATAGGGG 1925TGGTAGGGGTAGGAAGTGCAAGCTACAAAGTTATGACTCGTTCCAGCCATCAATCATTAGTCATAAAGTTAATGCCCAATATAACTCTCCTCAACAATTGCACGAGGGTAGGGATTGCAGAATACAGGAGACTACTGAGAACAGTTCTGGAACCAATTAGAGATGCACTTAATGCAATGACCCAGAATATAAGACCGGTTCAGAGTGTAGCTTCAAGTAGGAGACACAAGAGATTTGCGGGAGTTGTCCTGGCAGGTGCGGCCCTAGGCGTTGCCACAGCTGCTCAAATAACAGCCGGTATTGCACTTCACCAGTCCATGCTGAACTCTCAAGCCATCGACAATCTGAGAGCGAGCCTAGAAACTACTAATCAGGCAATTGAGGCAATCAGACAAGCAGGGCAGGAGATGATATTGGCTGTTCAGGGTGTCCAAGACTACATCAATAATGAGCTGATACCGTCTATGAATCAACTATCTTGTGATTTAATCGGCCAGAAGCTAGGGCTCAAATTGCTCAGATACTATACAGAAATCCTGTCATTATTTGGCCCCAGCTTACGGGACCCCATATCTGCGGAGATATCTATCCAGGCTTTGAGCTATGCGCTTGGAGGAGATATCAATAAGGTGTTGGAAAAGCTCGGATACAGTGGAGGTGATCTACTGGGCATCTTAGAGAGCAGAGGAATAAAGGCCCGGATAACTCACGTCGACACAGAGTCCTACTTCATTGTACTCAGTATAGCCTATCCGACGCTATCCGAGATTAAGGGGGTGATTGTCCACCGGCTAGAGGGGGTCTCGTACAACATAGGCTCTCAAGAGTGGTATACCACTGTGCCCAAGTATGTTGCAACCCAAGGGTACCTTATCTCGAATTTTGATGAGTCATCATGCACTTTCATGCCAGAGGGGACTGTGTGCAGCCAGAATGCCTTGTACCCGATGAGTCCTCTGCTCCAAGAATGCCTCCGGGGGTCCACTAAGTCCTGTGCTCGTACACTCGTATCCGGGTCTTTCGGGAACCGGTTCATTTTATCACAGGGGAACCTAATAGCCAATTGTGCATCAATCCTTTGCAAGTGTTACACAACAGGAACAATCATTAATCAAGACCCTGACAAGATCCTAACATACATTGCTGCCGATCACTGCCCGGTGGTCGAGGTGAATGGCGTGACCATCCAAGTCGGGAGCAGGAGGTATCCGGACGCTGTGTACTTGCACAGGATTGACCTCGGTCCTCCCATATCTTTGGAGAGGTTGGACGTAGGGACAAATCTGGGGAATGCAATTGCTAAGTTGGAGGATGCCAAGGAATTGTTGGAGTCATCGGACCAGATATTGAGGAGTATGAAAGGTTTATCGAGCACTAGTATAGTTTACATCCTGATTGCAGTGTGTCTTGGAGGATTGATAGGGATCCCCGCTTTAATATGTTGCTGCAGGGGGCGTTGTAACAAGAAGGGAGAACAAGTTGGTATGTCAAGACCAGGCCTAAAGCCTGATCTTACAGGAACATCAAAATCCTATGTAAGGTCACTCTGATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAATCTAGGC_H_MEASLES_B3 TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACT 41Sequence, NT (5′ CACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAA UTR, ORF, 3′GAAATATAAGAGCCACCATGTCACCGCAACGAGACCG UTR)GATAAATGCCTTCTACAAAGATAACCCTTATCCCAAGG Sequence Length:GAAGTAGGATAGTTATTAACAGAGAACATCTTATGATT 2065GACAGACCCTATGTTCTGCTGGCTGTTCTGTTCGTCATGTTTCTGAGCTTGATCGGATTGCTGGCAATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCGGAGATCCATAAAAGCCTCAGTACCAATCTGGATGTGACTAACTCCATCGAGCATCAGGTCAAGGACGTGCTGACACCACTCTTTAAAATCATCGGGGATGAAGTGGGCCTGAGAACACCTCAGAGATTCACTGACCTAGTGAAATTCATCTCGGACAAGATTAAATTCCTTAATCCGGATAGGGAGTACGACTTCAGAGATCTCACTTGGTGCATCAACCCGCCAGAGAGGATCAAACTAGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAGCTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGAACAACCACTCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGTCAATTCTCAAACATGTCGCTGTCCTTGTTGGACTTGTACTTAGGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTATGGGGGAACCTACCTAGTTGAAAAGCCTAATCTGAACAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGTACCGAGTGTTTGAAGTAGGTGTGATCAGAAACCCGGGTTTGGGGGCTCCGGTGTTCCATATGACAAACTATTTTGAGCAACCAGTCAGTAATGGTCTCGGCAACTGTATGGTGGCTTTGGGGGAGCTCAAACTCGCAGCCCTTTGTCACGGGGACGATTCTATCATAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTCGTCAAGCTGGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCTTATCAACGGATGATCCAGTGGTAGACAGGCTTTACCTCTCATCTCACAGAGGTGTCATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGAACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAAGGTAAAATCCAAGCACTCTGCGAGAATCCCGAGTGGGTACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCCTGTCTGTTGATCTGAGTCTGACGGTTGAGCTTAAAATCAAAATTGCTTCGGGATTCGGGCCATTGATCACACACGGCTCAGGGATGGACCTATACAAATCCAACTGCAACAATGTGTATTGGCTGACTATTCCGCCAATGAGAAATCTAGCCTTAGGCGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTCCCAATTAAGGAAGCAGGCGAAGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGACGGTGATGTCAAACTCAGTTCCAACCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTTTGGCAACCTACGATACCTCCAGGGTTGAGCATGCTGTGGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTATAAAGGGGGTCCCAATCGAACTACAAGTGGAATGCTTCACATGGGATCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCCGGTGGACTTATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCTACCCGGGAAGATGGAACCAATCGCAGATAATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG AGTGGGCGGC GC_H_MEASLES_B3ATGTCACCGCAACGAGACCGGATAAATGCCTTCTACAA 42 ORF Sequence, NTAGATAACCCTTATCCCAAGGGAAGTAGGATAGTTATTAACAGAGAACATCTTATGATTGACAGACCCTATGTTCTGCTGGCTGTTCTGTTCGTCATGTTTCTGAGCTTGATCGGATTGCTGGCAATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCGGAGATCCATAAAAGCCTCAGTACCAATCTGGATGTGACTAACTCCATCGAGCATCAGGTCAAGGACGTGCTGACACCACTCTTTAAAATCATCGGGGATGAAGTGGGCCTGAGAACACCTCAGAGATTCACTGACCTAGTGAAATTCATCTCGGACAAGATTAAATTCCTTAATCCGGATAGGGAGTACGACTTCAGAGATCTCACTTGGTGCATCAACCCGCCAGAGAGGATCAAACTAGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAGCTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGAACAACCACTCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGTCAATTCTCAAACATGTCGCTGTCCTTGTTGGACTTGTACTTAGGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTATGGGGGAACCTACCTAGTTGAAAAGCCTAATCTGAACAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGTACCGAGTGTTTGAAGTAGGTGTGATCAGAAACCCGGGTTTGGGGGCTCCGGTGTTCCATATGACAAACTATTTTGAGCAACCAGTCAGTAATGGTCTCGGCAACTGTATGGTGGCTTTGGGGGAGCTCAAACTCGCAGCCCTTTGTCACGGGGACGATTCTATCATAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTCGTCAAGCTGGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCTTATCAACGGATGATCCAGTGGTAGACAGGCTTTACCTCTCATCTCACAGAGGTGTCATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGAACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAAGGTAAAATCCAAGCACTCTGCGAGAATCCCGAGTGGGTACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCCTGTCTGTTGATCTGAGTCTGACGGTTGAGCTTAAAATCAAAATTGCTTCGGGATTCGGGCCATTGATCACACACGGCTCAGGGATGGACCTATACAAATCCAACTGCAACAATGTGTATTGGCTGACTATTCCGCCAATGAGAAATCTAGCCTTAGGCGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTCCCAATTAAGGAAGCAGGCGAAGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGACGGTGATGTCAAACTCAGTTCCAACCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTTTGGCAACCTACGATACCTCCAGGGTTGAGCATGCTGTGGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTATAAAGGGGGTCCCAATCGAACTACAAGTGGAATGCTTCACATGGGATCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCCGGTGGACTTATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCTACCCG GGAAGATGGAACCAATCGCAGATAAGC_H_MEASLES_B3 G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAT 43 mRNA SequenceATAAGAGCCACCATGTCACCGCAACGAGACCGGATAA (assumes T100 tail)ATGCCTTCTACAAAGATAACCCTTATCCCAAGGGAAGT Sequence Length:AGGATAGTTATTAACAGAGAACATCTTATGATTGACAG 2126ACCCTATGTTCTGCTGGCTGTTCTGTTCGTCATGTTTCTGAGCTTGATCGGATTGCTGGCAATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCGGAGATCCATAAAAGCCTCAGTACCAATCTGGATGTGACTAACTCCATCGAGCATCAGGTCAAGGACGTGCTGACACCACTCTTTAAAATCATCGGGGATGAAGTGGGCCTGAGAACACCTCAGAGATTCACTGACCTAGTGAAATTCATCTCGGACAAGATTAAATTCCTTAATCCGGATAGGGAGTACGACTTCAGAGATCTCACTTGGTGCATCAACCCGCCAGAGAGGATCAAACTAGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAGCTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGAACAACCACTCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGTCAATTCTCAAACATGTCGCTGTCCTTGTTGGACTTGTACTTAGGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTATGGGGGAACCTACCTAGTTGAAAAGCCTAATCTGAACAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGTACCGAGTGTTTGAAGTAGGTGTGATCAGAAACCCGGGTTTGGGGGCTCCGGTGTTCCATATGACAAACTATTTTGAGCAACCAGTCAGTAATGGTCTCGGCAACTGTATGGTGGCTTTGGGGGAGCTCAAACTCGCAGCCCTTTGTCACGGGGACGATTCTATCATAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTCGTCAAGCTGGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCTTATCAACGGATGATCCAGTGGTAGACAGGCTTTACCTCTCATCTCACAGAGGTGTCATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGAACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAAGGTAAAATCCAAGCACTCTGCGAGAATCCCGAGTGGGTACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCCTGTCTGTTGATCTGAGTCTGACGGTTGAGCTTAAAATCAAAATTGCTTCGGGATTCGGGCCATTGATCACACACGGCTCAGGGATGGACCTATACAAATCCAACTGCAACAATGTGTATTGGCTGACTATTCCGCCAATGAGAAATCTAGCCTTAGGCGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTCCCAATTAAGGAAGCAGGCGAAGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGACGGTGATGTCAAACTCAGTTCCAACCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTTTGGCAACCTACGATACCTCCAGGGTTGAGCATGCTGTGGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTATAAAGGGGGTCCCAATCGAACTACAAGTGGAATGCTTCACATGGGATCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCCGGTGGACTTATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCTACCCGGGAAGATGGAACCAATCGCAGATAATGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATC TAG GC_H_MEASLES_D8TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACT 44 Sequence, NT (5′CACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAA UTR, ORF, 3′GAAATATAAGAGCCACCATGTCACCACAACGAGACCG UTR)GATAAATGCCTTCTACAAAGACAACCCCCATCCTAAGG Sequence Length:GAAGTAGGATAGTTATTAACAGAGAACATCTTATGATT 2065GATAGACCTTATGTTTTGCTGGCTGTTCTATTCGTCATGTTTCTGAGCTTGATCGGGTTGCTAGCCATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCAGAGATCCATAAAAGCCTCAGCACCAATCTGGATGTAACTAACTCAATCGAGCATCAGGTTAAGGACGTGCTGACACCACTCTTCAAGATCATCGGTGATGAAGTGGGCTTGAGGACACCTCAGAGATTCACTGACCTAGTGAAGTTCATCTCTGACAAGATTAAATTCCTTAATCCGGACAGGGAATACGACTTCAGAGATCTCACTTGGTGTATCAACCCGCCAGAGAGAATCAAATTGGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAACTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGGGCAACCAATCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGCCAATTCTCAAACATGTCGCTGTCCCTGTTGGACTTGTATTTAAGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTACGGGGGAACTTACCTAGTGGAAAAGCCTAATCTGAGCAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGCACCGAGTGTTTGAAGTAGGTGTTATCAGAAATCCGGGTTTGGGGGCTCCGGTATTCCATATGACAAACTATCTTGAGCAACCAGTCAGTAATGATTTCAGCAACTGCATGGTGGCTTTGGGGGAGCTCAAGTTCGCAGCCCTCTGTCACAGGGAAGATTCTATCACAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTTGTCAAGCTAGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCCTATCAACGGATGATCCAGTGATAGACAGGCTTTACCTCTCATCTCACAGAGGCGTTATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGGACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAGGGTAAAATCCAAGCACTTTGCGAGAATCCCGAGTGGACACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCTTGTCTGTTGATCTGAGTCTGACAGTTGAGCTTAAAATCAAAATTGTTTCAGGATTCGGGCCATTGATCACACACGGTTCAGGGATGGACCTATACAAATCCAACCACAACAATATGTATTGGCTGACTATCCCGCCAATGAAGAACCTGGCCTTAGGTGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTTCCAATTAAGGAAGCAGGCGAGGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGATGGTGATGTCAAACTCAGTTCCAATCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTCTGGCAACCTACGATACTTCCAGAGTTGAACATGCTGTAGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTGTAAGGGGGGTCCCCATTGAATTACAAGTGGAATGCTTCACATGGGACCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCTGGTGGACATATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCCACTCGGGAAGATGGAACCAGCCGCAGATAGTGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAG TGGGCGGC GC_H_MEASLES_D8ATGTCACCACAACGAGACCGGATAAATGCCTTCTACAA 45 ORF Sequence, NTAGACAACCCCCATCCTAAGGGAAGTAGGATAGTTATTAACAGAGAACATCTTATGATTGATAGACCTTATGTTTTGCTGGCTGTTCTATTCGTCATGTTTCTGAGCTTGATCGGGTTGCTAGCCATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCAGAGATCCATAAAAGCCTCAGCACCAATCTGGATGTAACTAACTCAATCGAGCATCAGGTTAAGGACGTGCTGACACCACTCTTCAAGATCATCGGTGATGAAGTGGGCTTGAGGACACCTCAGAGATTCACTGACCTAGTGAAGTTCATCTCTGACAAGATTAAATTCCTTAATCCGGACAGGGAATACGACTTCAGAGATCTCACTTGGTGTATCAACCCGCCAGAGAGAATCAAATTGGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAACTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGGGCAACCAATCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGCCAATTCTCAAACATGTCGCTGTCCCTGTTGGACTTGTATTTAAGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTACGGGGGAACTTACCTAGTGGAAAAGCCTAATCTGAGCAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGCACCGAGTGTTTGAAGTAGGTGTTATCAGAAATCCGGGTTTGGGGGCTCCGGTATTCCATATGACAAACTATCTTGAGCAACCAGTCAGTAATGATTTCAGCAACTGCATGGTGGCTTTGGGGGAGCTCAAGTTCGCAGCCCTCTGTCACAGGGAAGATTCTATCACAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTTGTCAAGCTAGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCCTATCAACGGATGATCCAGTGATAGACAGGCTTTACCTCTCATCTCACAGAGGCGTTATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGGACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAGGGTAAAATCCAAGCACTTTGCGAGAATCCCGAGTGGACACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCTTGTCTGTTGATCTGAGTCTGACAGTTGAGCTTAAAATCAAAATTGTTTCAGGATTCGGGCCATTGATCACACACGGTTCAGGGATGGACCTATACAAATCCAACCACAACAATATGTATTGGCTGACTATCCCGCCAATGAAGAACCTGGCCTTAGGTGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTTCCAATTAAGGAAGCAGGCGAGGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGATGGTGATGTCAAACTCAGTTCCAATCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTCTGGCAACCTACGATACTTCCAGAGTTGAACATGCTGTAGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTGTAAGGGGGGTCCCCATTGAATTACAAGTGGAATGCTTCACATGGGACCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCTGGTGGACATATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCCACTCGGG AAGATGGAACCAGCCGCAGATAGGC_H_MEASLES_D8 G*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAT 46 mRNA SequenceATAAGAGCCACCATGTCACCACAACGAGACCGGATAA (assumes T100 tail)ATGCCTTCTACAAAGACAACCCCCATCCTAAGGGAAGT Sequence Length:AGGATAGTTATTAACAGAGAACATCTTATGATTGATAG 2126ACCTTATGTTTTGCTGGCTGTTCTATTCGTCATGTTTCTGAGCTTGATCGGGTTGCTAGCCATTGCAGGCATTAGACTTCATCGGGCAGCCATCTACACCGCAGAGATCCATAAAAGCCTCAGCACCAATCTGGATGTAACTAACTCAATCGAGCATCAGGTTAAGGACGTGCTGACACCACTCTTCAAGATCATCGGTGATGAAGTGGGCTTGAGGACACCTCAGAGATTCACTGACCTAGTGAAGTTCATCTCTGACAAGATTAAATTCCTTAATCCGGACAGGGAATACGACTTCAGAGATCTCACTTGGTGTATCAACCCGCCAGAGAGAATCAAATTGGATTATGATCAATACTGTGCAGATGTGGCTGCTGAAGAACTCATGAATGCATTGGTGAACTCAACTCTACTGGAGACCAGGGCAACCAATCAGTTCCTAGCTGTCTCAAAGGGAAACTGCTCAGGGCCCACTACAATCAGAGGCCAATTCTCAAACATGTCGCTGTCCCTGTTGGACTTGTATTTAAGTCGAGGTTACAATGTGTCATCTATAGTCACTATGACATCCCAGGGAATGTACGGGGGAACTTACCTAGTGGAAAAGCCTAATCTGAGCAGCAAAGGGTCAGAGTTGTCACAACTGAGCATGCACCGAGTGTTTGAAGTAGGTGTTATCAGAAATCCGGGTTTGGGGGCTCCGGTATTCCATATGACAAACTATCTTGAGCAACCAGTCAGTAATGATTTCAGCAACTGCATGGTGGCTTTGGGGGAGCTCAAGTTCGCAGCCCTCTGTCACAGGGAAGATTCTATCACAATTCCCTATCAGGGATCAGGGAAAGGTGTCAGCTTCCAGCTTGTCAAGCTAGGTGTCTGGAAATCCCCAACCGACATGCAATCCTGGGTCCCCCTATCAACGGATGATCCAGTGATAGACAGGCTTTACCTCTCATCTCACAGAGGCGTTATCGCTGACAATCAAGCAAAATGGGCTGTCCCGACAACACGGACAGATGACAAGTTGCGAATGGAGACATGCTTCCAGCAGGCGTGTAAGGGTAAAATCCAAGCACTTTGCGAGAATCCCGAGTGGACACCATTGAAGGATAACAGGATTCCTTCATACGGGGTCTTGTCTGTTGATCTGAGTCTGACAGTTGAGCTTAAAATCAAAATTGTTTCAGGATTCGGGCCATTGATCACACACGGTTCAGGGATGGACCTATACAAATCCAACCACAACAATATGTATTGGCTGACTATCCCGCCAATGAAGAACCTGGCCTTAGGTGTAATCAACACATTGGAGTGGATACCGAGATTCAAGGTTAGTCCCAACCTCTTCACTGTTCCAATTAAGGAAGCAGGCGAGGACTGCCATGCCCCAACATACCTACCTGCGGAGGTGGATGGTGATGTCAAACTCAGTTCCAATCTGGTGATTCTACCTGGTCAAGATCTCCAATATGTTCTGGCAACCTACGATACTTCCAGAGTTGAACATGCTGTAGTTTATTACGTTTACAGCCCAAGCCGCTCATTTTCTTACTTTTATCCTTTTAGGTTGCCTGTAAGGGGGGTCCCCATTGAATTACAAGTGGAATGCTTCACATGGGACCAAAAACTCTGGTGCCGTCACTTCTGTGTGCTTGCGGACTCAGAATCTGGTGGACATATCACTCACTCTGGGATGGTGGGCATGGGAGTCAGCTGCACAGCCACTCGGGAAGATGGAACCAGCCGCAGATAGTGATAATAGGCTGGAGCCTCGGTGGCCAAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAT CTAG MeV mRNA SequencesGC_F_MEASLES_B3.1 UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGAC 69Sequence, NT (5′ UCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAG UTR, ORF, 3′AAGAAAUAUAAGAGCCACCAUGGGUCUCAAGGUGAA UTR)CGUCUCUGCCGUAUUCAUGGCAGUACUGUUAACUCUC Sequence Length:CAAACACCCGCCGGUCAAAUUCAUUGGGGCAAUCUCU 1864CUAAGAUAGGGGUAGUAGGAAUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAAUUAAUGCCCAAUAUAACUCUCCUCAAUAACUGCACGAGGGUAGAGAUUGCAGAAUACAGGAGACUACUAAGAACAGUUUUGGAACCAAUUAGGGAUGCACUUAAUGCAAUGACCCAGAACAUAAGGCCGGUUCAGAGCGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUAGUCCUGGCAGGUGCGGCCCUAGGUGUUGCCACAGCUGCUCAGAUAACAGCCGGCAUUGCACUUCACCGGUCCAUGCUGAACUCUCAGGCCAUCGACAAUCUGAGAGCGAGCCUGGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAACCAGCUAUCUUGUGAUCUAAUCGGUCAGAAGCUCGGGCUCAAAUUGCUUAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCCUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGUUAUGCACUUGGAGGAGAUAUCAAUAAGGUGUUAGAAAAGCUCGGAUACAGUGGAGGCGAUUUACUAGGCAUCUUAGAGAGCAGAGGAAUAAAGGCUCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUAGUCCUCAGUAUAGCCUAUCCGACGCUGUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGUACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAAAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACCAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUUGGGAACCGGUUCAUUUUAUCACAAGGGAACCUAAUAGCCAAUUGUGCAUCAAUUCUUUGUAAGUGUUACACAACAGGUACGAUUAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCGCUGCCCGGUAGUCGAGGUGAACGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCAGACGCUGUGUACUUGCACAGAAUUGACCUCGGUCCUCCCAUAUCAUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCCAAAUUGGAGGAUGCCAAGGAAUUGUUGGAAUCAUCGGACCAGAUAUUGAGAAGUAUGAAAGGUUUAUCGAGCACUAGCAUAGUCUACAUCCUGAUUGCAGUGUGUCUUGGAGGGUUGAUAGGGAUCCCCACUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAAAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGACCUUACAGGAACAUCAAAAUCCUAUGUAAGAUCGCUUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC GC_F_MEASLES_B3.1AUGGGUCUCAAGGUGAACGUCUCUGCCGUAUUCAUGG 70 ORF Sequence, NTCAGUACUGUUAACUCUCCAAACACCCGCCGGUCAAAUUCAUUGGGGCAAUCUCUCUAAGAUAGGGGUAGUAGGAAUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAAUUAAUGCCCAAUAUAACUCUCCUCAAUAACUGCACGAGGGUAGAGAUUGCAGAAUACAGGAGACUACUAAGAACAGUUUUGGAACCAAUUAGGGAUGCACUUAAUGCAAUGACCCAGAACAUAAGGCCGGUUCAGAGCGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUAGUCCUGGCAGGUGCGGCCCUAGGUGUUGCCACAGCUGCUCAGAUAACAGCCGGCAUUGCACUUCACCGGUCCAUGCUGAACUCUCAGGCCAUCGACAAUCUGAGAGCGAGCCUGGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAACCAGCUAUCUUGUGAUCUAAUCGGUCAGAAGCUCGGGCUCAAAUUGCUUAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCCUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGUUAUGCACUUGGAGGAGAUAUCAAUAAGGUGUUAGAAAAGCUCGGAUACAGUGGAGGCGAUUUACUAGGCAUCUUAGAGAGCAGAGGAAUAAAGGCUCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUAGUCCUCAGUAUAGCCUAUCCGACGCUGUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGUACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAAAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACCAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUUGGGAACCGGUUCAUUUUAUCACAAGGGAACCUAAUAGCCAAUUGUGCAUCAAUUCUUUGUAAGUGUUACACAACAGGUACGAUUAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCGCUGCCCGGUAGUCGAGGUGAACGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCAGACGCUGUGUACUUGCACAGAAUUGACCUCGGUCCUCCCAUAUCAUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCCAAAUUGGAGGAUGCCAAGGAAUUGUUGGAAUCAUCGGACCAGAUAUUGAGAAGUAUGAAAGGUUUAUCGAGCACUAGCAUAGUCUACAUCCUGAUUGCAGUGUGUCUUGGAGGGUUGAUAGGGAUCCCCACUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAAAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGACCUUACAGGAACAUCAAAAUCCUAUGUAAGAUCGCUU UGA GC_F_MEASLES_B3.1G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 71 mRNA SequenceUAUAAGAGCCACCAUGGGUCUCAAGGUGAACGUCUCU (assumes T100 tail)GCCGUAUUCAUGGCAGUACUGUUAACUCUCCAAACAC mRNA SequenceCCGCCGGUCAAAUUCAUUGGGGCAAUCUCUCUAAGAU Length: 1925AGGGGUAGUAGGAAUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAAUUAAUGCCCAAUAUAACUCUCCUCAAUAACUGCACGAGGGUAGAGAUUGCAGAAUACAGGAGACUACUAAGAACAGUUUUGGAACCAAUUAGGGAUGCACUUAAUGCAAUGACCCAGAACAUAAGGCCGGUUCAGAGCGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUAGUCCUGGCAGGUGCGGCCCUAGGUGUUGCCACAGCUGCUCAGAUAACAGCCGGCAUUGCACUUCACCGGUCCAUGCUGAACUCUCAGGCCAUCGACAAUCUGAGAGCGAGCCUGGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAACCAGCUAUCUUGUGAUCUAAUCGGUCAGAAGCUCGGGCUCAAAUUGCUUAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCCUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGUUAUGCACUUGGAGGAGAUAUCAAUAAGGUGUUAGAAAAGCUCGGAUACAGUGGAGGCGAUUUACUAGGCAUCUUAGAGAGCAGAGGAAUAAAGGCUCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUAGUCCUCAGUAUAGCCUAUCCGACGCUGUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGUACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAAAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACCAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUUGGGAACCGGUUCAUUUUAUCACAAGGGAACCUAAUAGCCAAUUGUGCAUCAAUUCUUUGUAAGUGUUACACAACAGGUACGAUUAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCGCUGCCCGGUAGUCGAGGUGAACGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCAGACGCUGUGUACUUGCACAGAAUUGACCUCGGUCCUCCCAUAUCAUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCCAAAUUGGAGGAUGCCAAGGAAUUGUUGGAAUCAUCGGACCAGAUAUUGAGAAGUAUGAAAGGUUUAUCGAGCACUAGCAUAGUCUACAUCCUGAUUGCAGUGUGUCUUGGAGGGUUGAUAGGGAUCCCCACUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAAAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGACCUUACAGGAACAUCAAAAUCCUAUGUAAGAUCGCUUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAUCUAGGC_F_MEASLES_D8 UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGAC 72Sequence, NT (5′ UCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAG UTR, ORF, 3′AAGAAAUAUAAGAGCCACCAUGGGUCUCAAGGUGAA UTR)CGUCUCUGUCAUAUUCAUGGCAGUACUGUUAACUCUU Sequence Length:CAAACACCCACCGGUCAAAUCCAUUGGGGCAAUCUCU 1864CUAAGAUAGGGGUGGUAGGGGUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAGUUAAUGCCCAAUAUAACUCUCCUCAACAAUUGCACGAGGGUAGGGAUUGCAGAAUACAGGAGACUACUGAGAACAGUUCUGGAACCAAUUAGAGAUGCACUUAAUGCAAUGACCCAGAAUAUAAGACCGGUUCAGAGUGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUUGUCCUGGCAGGUGCGGCCCUAGGCGUUGCCACAGCUGCUCAAAUAACAGCCGGUAUUGCACUUCACCAGUCCAUGCUGAACUCUCAAGCCAUCGACAAUCUGAGAGCGAGCCUAGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAAUCAACUAUCUUGUGAUUUAAUCGGCCAGAAGCUAGGGCUCAAAUUGCUCAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCUUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGCUAUGCGCUUGGAGGAGAUAUCAAUAAGGUGUUGGAAAAGCUCGGAUACAGUGGAGGUGAUCUACUGGGCAUCUUAGAGAGCAGAGGAAUAAAGGCCCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUUGUACUCAGUAUAGCCUAUCCGACGCUAUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGCACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAGAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACUAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUCGGGAACCGGUUCAUUUUAUCACAGGGGAACCUAAUAGCCAAUUGUGCAUCAAUCCUUUGCAAGUGUUACACAACAGGAACAAUCAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCACUGCCCGGUGGUCGAGGUGAAUGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCGGACGCUGUGUACUUGCACAGGAUUGACCUCGGUCCUCCCAUAUCUUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCUAAGUUGGAGGAUGCCAAGGAAUUGUUGGAGUCAUCGGACCAGAUAUUGAGGAGUAUGAAAGGUUUAUCGAGCACUAGUAUAGUUUACAUCCUGAUUGCAGUGUGUCUUGGAGGAUUGAUAGGGAUCCCCGCUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAGAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGAUCUUACAGGAACAUCAAAAUCCUAUGUAAGGUCACUCUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC GC_F_MEASLES_D8AUGGGUCUCAAGGUGAACGUCUCUGUCAUAUUCAUG 73 ORF Sequence, NTGCAGUACUGUUAACUCUUCAAACACCCACCGGUCAAAUCCAUUGGGGCAAUCUCUCUAAGAUAGGGGUGGUAGGGGUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAGUUAAUGCCCAAUAUAACUCUCCUCAACAAUUGCACGAGGGUAGGGAUUGCAGAAUACAGGAGACUACUGAGAACAGUUCUGGAACCAAUUAGAGAUGCACUUAAUGCAAUGACCCAGAAUAUAAGACCGGUUCAGAGUGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUUGUCCUGGCAGGUGCGGCCCUAGGCGUUGCCACAGCUGCUCAAAUAACAGCCGGUAUUGCACUUCACCAGUCCAUGCUGAACUCUCAAGCCAUCGACAAUCUGAGAGCGAGCCUAGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAAUCAACUAUCUUGUGAUUUAAUCGGCCAGAAGCUAGGGCUCAAAUUGCUCAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCUUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGCUAUGCGCUUGGAGGAGAUAUCAAUAAGGUGUUGGAAAAGCUCGGAUACAGUGGAGGUGAUCUACUGGGCAUCUUAGAGAGCAGAGGAAUAAAGGCCCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUUGUACUCAGUAUAGCCUAUCCGACGCUAUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGCACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAGAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACUAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUCGGGAACCGGUUCAUUUUAUCACAGGGGAACCUAAUAGCCAAUUGUGCAUCAAUCCUUUGCAAGUGUUACACAACAGGAACAAUCAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCACUGCCCGGUGGUCGAGGUGAAUGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCGGACGCUGUGUACUUGCACAGGAUUGACCUCGGUCCUCCCAUAUCUUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCUAAGUUGGAGGAUGCCAAGGAAUUGUUGGAGUCAUCGGACCAGAUAUUGAGGAGUAUGAAAGGUUUAUCGAGCACUAGUAUAGUUUACAUCCUGAUUGCAGUGUGUCUUGGAGGAUUGAUAGGGAUCCCCGCUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAGAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGAUCUUACAGGAACAUCAAAAUCCUAUGUAAGGUC ACUCUGA GC_F_MEASLES_D8G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 74 mRNA SequenceUAUAAGAGCCACCAUGGGUCUCAAGGUGAACGUCUCU (assumes T100 tail)GUCAUAUUCAUGGCAGUACUGUUAACUCUUCAAACAC Sequence Length:CCACCGGUCAAAUCCAUUGGGGCAAUCUCUCUAAGAU 1925AGGGGUGGUAGGGGUAGGAAGUGCAAGCUACAAAGUUAUGACUCGUUCCAGCCAUCAAUCAUUAGUCAUAAAGUUAAUGCCCAAUAUAACUCUCCUCAACAAUUGCACGAGGGUAGGGAUUGCAGAAUACAGGAGACUACUGAGAACAGUUCUGGAACCAAUUAGAGAUGCACUUAAUGCAAUGACCCAGAAUAUAAGACCGGUUCAGAGUGUAGCUUCAAGUAGGAGACACAAGAGAUUUGCGGGAGUUGUCCUGGCAGGUGCGGCCCUAGGCGUUGCCACAGCUGCUCAAAUAACAGCCGGUAUUGCACUUCACCAGUCCAUGCUGAACUCUCAAGCCAUCGACAAUCUGAGAGCGAGCCUAGAAACUACUAAUCAGGCAAUUGAGGCAAUCAGACAAGCAGGGCAGGAGAUGAUAUUGGCUGUUCAGGGUGUCCAAGACUACAUCAAUAAUGAGCUGAUACCGUCUAUGAAUCAACUAUCUUGUGAUUUAAUCGGCCAGAAGCUAGGGCUCAAAUUGCUCAGAUACUAUACAGAAAUCCUGUCAUUAUUUGGCCCCAGCUUACGGGACCCCAUAUCUGCGGAGAUAUCUAUCCAGGCUUUGAGCUAUGCGCUUGGAGGAGAUAUCAAUAAGGUGUUGGAAAAGCUCGGAUACAGUGGAGGUGAUCUACUGGGCAUCUUAGAGAGCAGAGGAAUAAAGGCCCGGAUAACUCACGUCGACACAGAGUCCUACUUCAUUGUACUCAGUAUAGCCUAUCCGACGCUAUCCGAGAUUAAGGGGGUGAUUGUCCACCGGCUAGAGGGGGUCUCGUACAACAUAGGCUCUCAAGAGUGGUAUACCACUGUGCCCAAGUAUGUUGCAACCCAAGGGUACCUUAUCUCGAAUUUUGAUGAGUCAUCAUGCACUUUCAUGCCAGAGGGGACUGUGUGCAGCCAGAAUGCCUUGUACCCGAUGAGUCCUCUGCUCCAAGAAUGCCUCCGGGGGUCCACUAAGUCCUGUGCUCGUACACUCGUAUCCGGGUCUUUCGGGAACCGGUUCAUUUUAUCACAGGGGAACCUAAUAGCCAAUUGUGCAUCAAUCCUUUGCAAGUGUUACACAACAGGAACAAUCAUUAAUCAAGACCCUGACAAGAUCCUAACAUACAUUGCUGCCGAUCACUGCCCGGUGGUCGAGGUGAAUGGCGUGACCAUCCAAGUCGGGAGCAGGAGGUAUCCGGACGCUGUGUACUUGCACAGGAUUGACCUCGGUCCUCCCAUAUCUUUGGAGAGGUUGGACGUAGGGACAAAUCUGGGGAAUGCAAUUGCUAAGUUGGAGGAUGCCAAGGAAUUGUUGGAGUCAUCGGACCAGAUAUUGAGGAGUAUGAAAGGUUUAUCGAGCACUAGUAUAGUUUACAUCCUGAUUGCAGUGUGUCUUGGAGGAUUGAUAGGGAUCCCCGCUUUAAUAUGUUGCUGCAGGGGGCGUUGUAACAAGAAGGGAGAACAAGUUGGUAUGUCAAGACCAGGCCUAAAGCCUGAUCUUACAGGAACAUCAAAAUCCUAUGUAAGGUCACUCUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAUCUAGGC_H_MEASLES_B3 UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGAC 75Sequence, NT (5′ UCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAG UTR, ORF, 3′AAGAAAUAUAAGAGCCACCAUGUCACCGCAACGAGAC UTR)CGGAUAAAUGCCUUCUACAAAGAUAACCCUUAUCCCA Sequence Length:AGGGAAGUAGGAUAGUUAUUAACAGAGAACAUCUUA 2065UGAUUGACAGACCCUAUGUUCUGCUGGCUGUUCUGUUCGUCAUGUUUCUGAGCUUGAUCGGAUUGCUGGCAAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCGGAGAUCCAUAAAAGCCUCAGUACCAAUCUGGAUGUGACUAACUCCAUCGAGCAUCAGGUCAAGGACGUGCUGACACCACUCUUUAAAAUCAUCGGGGAUGAAGUGGGCCUGAGAACACCUCAGAGAUUCACUGACCUAGUGAAAUUCAUCUCGGACAAGAUUAAAUUCCUUAAUCCGGAUAGGGAGUACGACUUCAGAGAUCUCACUUGGUGCAUCAACCCGCCAGAGAGGAUCAAACUAGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAGCUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGAACAACCACUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGUCAAUUCUCAAACAUGUCGCUGUCCUUGUUGGACUUGUACUUAGGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUAUGGGGGAACCUACCUAGUUGAAAAGCCUAAUCUGAACAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGUACCGAGUGUUUGAAGUAGGUGUGAUCAGAAACCCGGGUUUGGGGGCUCCGGUGUUCCAUAUGACAAACUAUUUUGAGCAACCAGUCAGUAAUGGUCUCGGCAACUGUAUGGUGGCUUUGGGGGAGCUCAAACUCGCAGCCCUUUGUCACGGGGACGAUUCUAUCAUAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUCGUCAAGCUGGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCUUAUCAACGGAUGAUCCAGUGGUAGACAGGCUUUACCUCUCAUCUCACAGAGGUGUCAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGAACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAAGGUAAAAUCCAAGCACUCUGCGAGAAUCCCGAGUGGGUACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCCUGUCUGUUGAUCUGAGUCUGACGGUUGAGCUUAAAAUCAAAAUUGCUUCGGGAUUCGGGCCAUUGAUCACACACGGCUCAGGGAUGGACCUAUACAAAUCCAACUGCAACAAUGUGUAUUGGCUGACUAUUCCGCCAAUGAGAAAUCUAGCCUUAGGCGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUCCCAAUUAAGGAAGCAGGCGAAGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGACGGUGAUGUCAAACUCAGUUCCAACCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUUUGGCAACCUACGAUACCUCCAGGGUUGAGCAUGCUGUGGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUAUAAAGGGGGUCCCAAUCGAACUACAAGUGGAAUGCUUCACAUGGGAUCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCCGGUGGACUUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCUACCCGGGAAGAUGGAACCAAUCGCAGAUAAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGC GC_H_MEASLES_B3AUGUCACCGCAACGAGACCGGAUAAAUGCCUUCUACA 76 ORF Sequence, NTAAGAUAACCCUUAUCCCAAGGGAAGUAGGAUAGUUAUUAACAGAGAACAUCUUAUGAUUGACAGACCCUAUGUUCUGCUGGCUGUUCUGUUCGUCAUGUUUCUGAGCUUGAUCGGAUUGCUGGCAAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCGGAGAUCCAUAAAAGCCUCAGUACCAAUCUGGAUGUGACUAACUCCAUCGAGCAUCAGGUCAAGGACGUGCUGACACCACUCUUUAAAAUCAUCGGGGAUGAAGUGGGCCUGAGAACACCUCAGAGAUUCACUGACCUAGUGAAAUUCAUCUCGGACAAGAUUAAAUUCCUUAAUCCGGAUAGGGAGUACGACUUCAGAGAUCUCACUUGGUGCAUCAACCCGCCAGAGAGGAUCAAACUAGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAGCUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGAACAACCACUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGUCAAUUCUCAAACAUGUCGCUGUCCUUGUUGGACUUGUACUUAGGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUAUGGGGGAACCUACCUAGUUGAAAAGCCUAAUCUGAACAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGUACCGAGUGUUUGAAGUAGGUGUGAUCAGAAACCCGGGUUUGGGGGCUCCGGUGUUCCAUAUGACAAACUAUUUUGAGCAACCAGUCAGUAAUGGUCUCGGCAACUGUAUGGUGGCUUUGGGGGAGCUCAAACUCGCAGCCCUUUGUCACGGGGACGAUUCUAUCAUAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUCGUCAAGCUGGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCUUAUCAACGGAUGAUCCAGUGGUAGACAGGCUUUACCUCUCAUCUCACAGAGGUGUCAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGAACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAAGGUAAAAUCCAAGCACUCUGCGAGAAUCCCGAGUGGGUACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCCUGUCUGUUGAUCUGAGUCUGACGGUUGAGCUUAAAAUCAAAAUUGCUUCGGGAUUCGGGCCAUUGAUCACACACGGCUCAGGGAUGGACCUAUACAAAUCCAACUGCAACAAUGUGUAUUGGCUGACUAUUCCGCCAAUGAGAAAUCUAGCCUUAGGCGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUCCCAAUUAAGGAAGCAGGCGAAGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGACGGUGAUGUCAAACUCAGUUCCAACCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUUUGGCAACCUACGAUACCUCCAGGGUUGAGCAUGCUGUGGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUAUAAAGGGGGUCCCAAUCGAACUACAAGUGGAAUGCUUCACAUGGGAUCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCCGGUGGACUUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCUACCCGGGAAG AUGGAACCAAUCGCAGAUAAGC_H_MEASLES_B3 G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 77 mRNA SequenceUAUAAGAGCCACCAUGUCACCGCAACGAGACCGGAUA (assumes T100 Tail)AAUGCCUUCUACAAAGAUAACCCUUAUCCCAAGGGAA Sequence Length:GUAGGAUAGUUAUUAACAGAGAACAUCUUAUGAUUG 2126ACAGACCCUAUGUUCUGCUGGCUGUUCUGUUCGUCAUGUUUCUGAGCUUGAUCGGAUUGCUGGCAAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCGGAGAUCCAUAAAAGCCUCAGUACCAAUCUGGAUGUGACUAACUCCAUCGAGCAUCAGGUCAAGGACGUGCUGACACCACUCUUUAAAAUCAUCGGGGAUGAAGUGGGCCUGAGAACACCUCAGAGAUUCACUGACCUAGUGAAAUUCAUCUCGGACAAGAUUAAAUUCCUUAAUCCGGAUAGGGAGUACGACUUCAGAGAUCUCACUUGGUGCAUCAACCCGCCAGAGAGGAUCAAACUAGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAGCUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGAACAACCACUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGUCAAUUCUCAAACAUGUCGCUGUCCUUGUUGGACUUGUACUUAGGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUAUGGGGGAACCUACCUAGUUGAAAAGCCUAAUCUGAACAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGUACCGAGUGUUUGAAGUAGGUGUGAUCAGAAACCCGGGUUUGGGGGCUCCGGUGUUCCAUAUGACAAACUAUUUUGAGCAACCAGUCAGUAAUGGUCUCGGCAACUGUAUGGUGGCUUUGGGGGAGCUCAAACUCGCAGCCCUUUGUCACGGGGACGAUUCUAUCAUAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUCGUCAAGCUGGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCUUAUCAACGGAUGAUCCAGUGGUAGACAGGCUUUACCUCUCAUCUCACAGAGGUGUCAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGAACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAAGGUAAAAUCCAAGCACUCUGCGAGAAUCCCGAGUGGGUACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCCUGUCUGUUGAUCUGAGUCUGACGGUUGAGCUUAAAAUCAAAAUUGCUUCGGGAUUCGGGCCAUUGAUCACACACGGCUCAGGGAUGGACCUAUACAAAUCCAACUGCAACAAUGUGUAUUGGCUGACUAUUCCGCCAAUGAGAAAUCUAGCCUUAGGCGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUCCCAAUUAAGGAAGCAGGCGAAGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGACGGUGAUGUCAAACUCAGUUCCAACCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUUUGGCAACCUACGAUACCUCCAGGGUUGAGCAUGCUGUGGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUAUAAAGGGGGUCCCAAUCGAACUACAAGUGGAAUGCUUCACAUGGGAUCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCCGGUGGACUUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCUACCCGGGAAGAUGGAACCAAUCGCAGAUAAUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGGC_H_MEASLES_D8 UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGAC 78Sequence, NT (5′ UCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAG UTR, ORF, 3′AAGAAAUAUAAGAGCCACCAUGUCACCACAACGAGAC UTR)CGGAUAAAUGCCUUCUACAAAGACAACCCCCAUCCUA Sequence Length:AGGGAAGUAGGAUAGUUAUUAACAGAGAACAUCUUA 2065UGAUUGAUAGACCUUAUGUUUUGCUGGCUGUUCUAUUCGUCAUGUUUCUGAGCUUGAUCGGGUUGCUAGCCAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCAGAGAUCCAUAAAAGCCUCAGCACCAAUCUGGAUGUAACUAACUCAAUCGAGCAUCAGGUUAAGGACGUGCUGACACCACUCUUCAAGAUCAUCGGUGAUGAAGUGGGCUUGAGGACACCUCAGAGAUUCACUGACCUAGUGAAGUUCAUCUCUGACAAGAUUAAAUUCCUUAAUCCGGACAGGGAAUACGACUUCAGAGAUCUCACUUGGUGUAUCAACCCGCCAGAGAGAAUCAAAUUGGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAACUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGGGCAACCAAUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGCCAAUUCUCAAACAUGUCGCUGUCCCUGUUGGACUUGUAUUUAAGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUACGGGGGAACUUACCUAGUGGAAAAGCCUAAUCUGAGCAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGCACCGAGUGUUUGAAGUAGGUGUUAUCAGAAAUCCGGGUUUGGGGGCUCCGGUAUUCCAUAUGACAAACUAUCUUGAGCAACCAGUCAGUAAUGAUUUCAGCAACUGCAUGGUGGCUUUGGGGGAGCUCAAGUUCGCAGCCCUCUGUCACAGGGAAGAUUCUAUCACAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUUGUCAAGCUAGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCCUAUCAACGGAUGAUCCAGUGAUAGACAGGCUUUACCUCUCAUCUCACAGAGGCGUUAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGGACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAGGGUAAAAUCCAAGCACUUUGCGAGAAUCCCGAGUGGACACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCUUGUCUGUUGAUCUGAGUCUGACAGUUGAGCUUAAAAUCAAAAUUGUUUCAGGAUUCGGGCCAUUGAUCACACACGGUUCAGGGAUGGACCUAUACAAAUCCAACCACAACAAUAUGUAUUGGCUGACUAUCCCGCCAAUGAAGAACCUGGCCUUAGGUGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUUCCAAUUAAGGAAGCAGGCGAGGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGAUGGUGAUGUCAAACUCAGUUCCAAUCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUCUGGCAACCUACGAUACUUCCAGAGUUGAACAUGCUGUAGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUGUAAGGGGGGUCCCCAUUGAAUUACAAGUGGAAUGCUUCACAUGGGACCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCUGGUGGACAUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCCACUCGGGAAGAUGGAACCAGCCGCAGAUAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGU GGGCGGC GC_H_MEASLES_D8AUGUCACCACAACGAGACCGGAUAAAUGCCUUCUACA 79 ORF Sequence, NTAAGACAACCCCCAUCCUAAGGGAAGUAGGAUAGUUAUUAACAGAGAACAUCUUAUGAUUGAUAGACCUUAUGUUUUGCUGGCUGUUCUAUUCGUCAUGUUUCUGAGCUUGAUCGGGUUGCUAGCCAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCAGAGAUCCAUAAAAGCCUCAGCACCAAUCUGGAUGUAACUAACUCAAUCGAGCAUCAGGUUAAGGACGUGCUGACACCACUCUUCAAGAUCAUCGGUGAUGAAGUGGGCUUGAGGACACCUCAGAGAUUCACUGACCUAGUGAAGUUCAUCUCUGACAAGAUUAAAUUCCUUAAUCCGGACAGGGAAUACGACUUCAGAGAUCUCACUUGGUGUAUCAACCCGCCAGAGAGAAUCAAAUUGGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAACUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGGGCAACCAAUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGCCAAUUCUCAAACAUGUCGCUGUCCCUGUUGGACUUGUAUUUAAGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUACGGGGGAACUUACCUAGUGGAAAAGCCUAAUCUGAGCAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGCACCGAGUGUUUGAAGUAGGUGUUAUCAGAAAUCCGGGUUUGGGGGCUCCGGUAUUCCAUAUGACAAACUAUCUUGAGCAACCAGUCAGUAAUGAUUUCAGCAACUGCAUGGUGGCUUUGGGGGAGCUCAAGUUCGCAGCCCUCUGUCACAGGGAAGAUUCUAUCACAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUUGUCAAGCUAGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCCUAUCAACGGAUGAUCCAGUGAUAGACAGGCUUUACCUCUCAUCUCACAGAGGCGUUAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGGACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAGGGUAAAAUCCAAGCACUUUGCGAGAAUCCCGAGUGGACACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCUUGUCUGUUGAUCUGAGUCUGACAGUUGAGCUUAAAAUCAAAAUUGUUUCAGGAUUCGGGCCAUUGAUCACACACGGUUCAGGGAUGGACCUAUACAAAUCCAACCACAACAAUAUGUAUUGGCUGACUAUCCCGCCAAUGAAGAACCUGGCCUUAGGUGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUUCCAAUUAAGGAAGCAGGCGAGGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGAUGGUGAUGUCAAACUCAGUUCCAAUCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUCUGGCAACCUACGAUACUUCCAGAGUUGAACAUGCUGUAGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUGUAAGGGGGGUCCCCAUUGAAUUACAAGUGGAAUGCUUCACAUGGGACCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCUGGUGGACAUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCCACUCGGGAAGAU GGAACCAGCCGCAGAUAG GC_H_MEASLES_D8G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 80 mRNA SequenceUAUAAGAGCCACCAUGUCACCACAACGAGACCGGAUA (assumes T100 tail)AAUGCCUUCUACAAAGACAACCCCCAUCCUAAGGGAA Sequence Length:GUAGGAUAGUUAUUAACAGAGAACAUCUUAUGAUUG 2126AUAGACCUUAUGUUUUGCUGGCUGUUCUAUUCGUCAUGUUUCUGAGCUUGAUCGGGUUGCUAGCCAUUGCAGGCAUUAGACUUCAUCGGGCAGCCAUCUACACCGCAGAGAUCCAUAAAAGCCUCAGCACCAAUCUGGAUGUAACUAACUCAAUCGAGCAUCAGGUUAAGGACGUGCUGACACCACUCUUCAAGAUCAUCGGUGAUGAAGUGGGCUUGAGGACACCUCAGAGAUUCACUGACCUAGUGAAGUUCAUCUCUGACAAGAUUAAAUUCCUUAAUCCGGACAGGGAAUACGACUUCAGAGAUCUCACUUGGUGUAUCAACCCGCCAGAGAGAAUCAAAUUGGAUUAUGAUCAAUACUGUGCAGAUGUGGCUGCUGAAGAACUCAUGAAUGCAUUGGUGAACUCAACUCUACUGGAGACCAGGGCAACCAAUCAGUUCCUAGCUGUCUCAAAGGGAAACUGCUCAGGGCCCACUACAAUCAGAGGCCAAUUCUCAAACAUGUCGCUGUCCCUGUUGGACUUGUAUUUAAGUCGAGGUUACAAUGUGUCAUCUAUAGUCACUAUGACAUCCCAGGGAAUGUACGGGGGAACUUACCUAGUGGAAAAGCCUAAUCUGAGCAGCAAAGGGUCAGAGUUGUCACAACUGAGCAUGCACCGAGUGUUUGAAGUAGGUGUUAUCAGAAAUCCGGGUUUGGGGGCUCCGGUAUUCCAUAUGACAAACUAUCUUGAGCAACCAGUCAGUAAUGAUUUCAGCAACUGCAUGGUGGCUUUGGGGGAGCUCAAGUUCGCAGCCCUCUGUCACAGGGAAGAUUCUAUCACAAUUCCCUAUCAGGGAUCAGGGAAAGGUGUCAGCUUCCAGCUUGUCAAGCUAGGUGUCUGGAAAUCCCCAACCGACAUGCAAUCCUGGGUCCCCCUAUCAACGGAUGAUCCAGUGAUAGACAGGCUUUACCUCUCAUCUCACAGAGGCGUUAUCGCUGACAAUCAAGCAAAAUGGGCUGUCCCGACAACACGGACAGAUGACAAGUUGCGAAUGGAGACAUGCUUCCAGCAGGCGUGUAAGGGUAAAAUCCAAGCACUUUGCGAGAAUCCCGAGUGGACACCAUUGAAGGAUAACAGGAUUCCUUCAUACGGGGUCUUGUCUGUUGAUCUGAGUCUGACAGUUGAGCUUAAAAUCAAAAUUGUUUCAGGAUUCGGGCCAUUGAUCACACACGGUUCAGGGAUGGACCUAUACAAAUCCAACCACAACAAUAUGUAUUGGCUGACUAUCCCGCCAAUGAAGAACCUGGCCUUAGGUGUAAUCAACACAUUGGAGUGGAUACCGAGAUUCAAGGUUAGUCCCAACCUCUUCACUGUUCCAAUUAAGGAAGCAGGCGAGGACUGCCAUGCCCCAACAUACCUACCUGCGGAGGUGGAUGGUGAUGUCAAACUCAGUUCCAAUCUGGUGAUUCUACCUGGUCAAGAUCUCCAAUAUGUUCUGGCAACCUACGAUACUUCCAGAGUUGAACAUGCUGUAGUUUAUUACGUUUACAGCCCAAGCCGCUCAUUUUCUUACUUUUAUCCUUUUAGGUUGCCUGUAAGGGGGGUCCCCAUUGAAUUACAAGUGGAAUGCUUCACAUGGGACCAAAAACUCUGGUGCCGUCACUUCUGUGUGCUUGCGGACUCAGAAUCUGGUGGACAUAUCACUCACUCUGGGAUGGUGGGCAUGGGAGUCAGCUGCACAGCCACUCGGGAAGAUGGAACCAGCCGCAGAUAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG

TABLE 14 MeV Amino Acid Sequences SEQ ID Description Sequence NO:GC_F_MEASLES_B3.1 MGLKVNVSAVFMAVLLTLQTPAGQIHWGNLSKIGVVG 47ORF Sequence, AA IGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHRSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYFIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADRCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPISLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLIGIPTLICCCR GRCNKKGEQVGMSRPGLKPDLTGTSKSYVRSL*GC_F_MEASLES_D8 MGLKVNVSVIFMAVLLTLQTPTGQIHWGNLSKIGVVG 48ORF Sequence, AA VGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVGIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYFIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPISLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLIGIPALICCCR GRCNKKGEQVGMSRPGLKPDLTGTSKSYVRSL*GC_H_MEASLES_B3 MSPQRDRINAFYKDNPYPKGSRIVINREHLMIDRPYV 49ORF Sequence, AA LLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRTTTQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLNSKGSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYFEQPVSNGLGNCMVALGELKLAALCHGDDSIIIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVVDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWVPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNCNNVYWLTIPPMRNLALGVINTLEWIPRFKVSPNLFTVPIKEAGEDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGG LITHSGMVGMGVSCTATREDGTNRR*GC_H_MEASLES_D8 MSPQRDRINAFYKDNPHPKGSRIVINREHLMIDRPYV 50ORF Sequence, AA LLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAEELMNALVNSTLLETRATNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLSRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKGSELSQLSMHRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDFSNCMVALGELKFAALCHREDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWTPLKDNRIPSYGVLSVDLSLTVELKIKIVSGFGPLITHGSGMDLYKSNHNNMYWLTIPPMKNLALGVINTLEWIPRFKVSPNLFTVPIKEAGEDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPVRGVPIELQVECFTWDQKLWCRHFCVLADSESGG HITHSGMVGMGVSCTATREDGTSRR*

TABLE 15 MeV NCBI Accession Numbers (Amino Acid Sequences) Type VirusName GenBank Accession hemagglutinin hemagglutinin [Measles virus strainMoraten] AAF85673.1 hemagglutinin hemagglutinin [Measles virus strainRubeovax] AAF85689.1 hemagglutinin hemagglutinin [Measles virus]AAF89824.1 hemagglutinin hemagglutinin protein [Measles virus]CAA91369.1 hemagglutinin hemagglutinin [Measles virus] BAJ23068.1hemagglutinin hemagglutinin protein [Measles virus] BAB39848.1hemagglutinin hemagglutinin [Measles virus] AAA50551.1 hemagglutininRecName: Full = Hemagglutinin glycoprotein P08362.1 hemagglutininhemagglutinin [Measles virus] AAB63802.1 hemagglutinin hemagglutinin[Measles virus] AAA56650.1 hemagglutinin hemagglutinin [Measles virus]AAA56642.1 hemagglutinin hemagglutinin [Measles virus] AAA74936.1hemagglutinin hemagglutinin protein [Measles virus] BAH56665.1hemagglutinin hemagglutinin [Measles virus] ACC86105.1 hemagglutininhemagglutinin [Measles virus strain Edmonston-Zagreb] AAF85697.1hemagglutinin hemagglutinin [Measles virus] AAR89413.1 hemagglutininhemagglutinin [Measles virus] AAA56653.1 hemagglutinin RecName: Full =Hemagglutinin glycoprotein P35971.1 hemagglutinin Hemagglutinin [Measlesvirus] CAB94916.1 hemagglutinin hemagglutinin [Measles virus] AAC03036.1hemagglutinin hemagglutinin [Measles virus] AAF85681.1 hemagglutininHemagglutinin [Measles virus] CAB94927.1 hemagglutinin Hemagglutinin[Measles virus] CAB94925.1 hemagglutinin hemagglutinin protein [Measlesvirus] BAB39835.1 hemagglutinin Hemagglutinin [Measles virus] CAB94931.1hemagglutinin hemagglutinin [Measles virus genotype A] AFO84712.1hemagglutinin hemagglutinin [Measles virus] AAA56639.1 hemagglutininHemagglutinin [Measles virus] CAB94926.1 hemagglutinin hemagglutininprotein [Measles virus] BAB39836.1 hemagglutinin Hemagglutinin [Measlesvirus] CAB94929.1 hemagglutinin RecName: Full = Hemagglutininglycoprotein P06830.1 hemagglutinin Hemagglutinin [Measles virus]CAB94928.1 hemagglutinin hemagglutinin protein [Measles virus]BAB39837.1 hemagglutinin hemagglutinin [Measles virus] AAA74935.1hemagglutinin hemagglutinin protein [Measles virus] CAB43780.1hemagglutinin hemagglutinin [Measles virus] BAA09952.1 hemagglutininhemagglutinin protein [Measles virus] CAB43815.1 hemagglutininhemagglutinin [Measles virus] AAF28390.1 hemagglutinin Hemagglutinin[Measles virus] CAB94923.1 hemagglutinin hemagglutinin protein [Measlesvirus] CAB43785.1 hemagglutinin hemagglutinin [Measles virus] ABD34001.1hemagglutinin hemagglutinin protein [Measles virus] CAB43782.1hemagglutinin hemagglutinin protein [Measles virus] CAB43781.1hemagglutinin hemagglutinin [Measles virus] BAH22353.1 hemagglutininhemagglutinin [Measles virus] AAC35878.2 hemagglutinin hemagglutininprotein [Measles virus] AAL86996.1 hemagglutinin hemagglutinin [Measlesvirus] CAA76066.2 hemagglutinin hemagglutinin [Measles virus] AAA46428.1hemagglutinin hemagglutinin protein [Measles virus] CAB43803.1hemagglutinin Hemagglutinin [Measles virus] CAB94918.1 hemagglutininhemagglutinin [Measles virus] AAF72162.1 hemagglutinin hemagglutinin[Measles virus] AAM70154.1 hemagglutinin hemagglutinin protein [Measlesvirus] CAB43776.1 hemagglutinin hemagglutinin [Measles virus genotypeD4] ACT78395.1 hemagglutinin hemagglutinin [Measles virus genotype D7]AAL02030.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43789.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43774.1 hemagglutinin Hemagglutinin [Measles virus] CAB94920.1hemagglutinin Hemagglutinin [Measles virus] CAB94922.1 hemagglutininhemagglutinin [Measles virus] ABB59491.1 hemagglutinin hemagglutininprotein [Measles virus] BAB39843.1 hemagglutinin hemagglutinin protein[Measles virus] CAB43804.1 hemagglutinin hemagglutinin [Measles virus]AAX52048.1 hemagglutinin Hemagglutinin [Measles virus] CAB94930.1hemagglutinin hemagglutinin [Measles virus] AAA74526.1 hemagglutininhemagglutinin protein [Measles virus] CAB43814.1 hemagglutininhemagglutinin [Measles virus] ABB59493.1 hemagglutinin hemagglutinin[Measles virus genotype D4] AAL02019.1 hemagglutinin Hemagglutinin[Measles virus] CAB94919.1 hemagglutinin hemagglutinin protein [Measlesvirus] AAL86997.1 hemagglutinin hemagglutinin [Measles virus genotypeC2] AAL02017.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43769.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43808.1 hemagglutinin hemagglutinin [Measles virus] BAO97032.1hemagglutinin hemagglutinin protein [Measles virus] CAB43805.1hemagglutinin hemagglutinin protein [Measles virus] CAB43777.1hemagglutinin hemagglutinin [Measles virus] AAL67793.1 hemagglutininhemagglutinin [Measles virus] AAF89816.1 hemagglutinin hemagglutinin[Measles virus genotype D4] AAL02020.1 hemagglutinin hemagglutininprotein [Measles virus] CAB43786.1 hemagglutinin hemagglutinin protein[Measles virus strain AEP40452.1 MVi/New Jersey.USA/45.05] hemagglutininhemagglutinin [Measles virus] AAA74531.1 hemagglutinin hemagglutinin[Measles virus] AAB63800.1 hemagglutinin hemagglutinin [Measles virus]AAO21711.1 hemagglutinin hemagglutinin [Measles virus genotype D8]ALE27189.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43810.1 hemagglutinin hemagglutinin [Measles virus] AAF89817.1hemagglutinin hemagglutinin [Measles virus genotype D6] AAL02022.1hemagglutinin hemagglutinin protein [Measles virus] CAB43800.1hemagglutinin hemagglutinin protein [Measles virus genotype B3]AGA17219.1 hemagglutinin hemagglutinin protein [Measles virus]CAB43770.1 hemagglutinin hemagglutinin protein [Measles virus strainAEP40444.1 MVi/Texas.USA/4.07] hemagglutinin hemagglutinin [Measlesvirus] AAX52047.1 hemagglutinin hemagglutinin [Measles virus] AAB63794.1hemagglutinin hemagglutinin [Measles virus] AAB63796.1 hemagglutininhemagglutinin [Measles virus] AAA74528.1 hemagglutinin hemagglutinin[Measles virus] AAB63774.1 hemagglutinin hemagglutinin [Measles virus]AAB63795.1 hemagglutinin hemagglutinin [Measles virus] AAA74519.1hemagglutinin hemagglutinin protein [Measles virus] CAB43778.1 fusionprotein fusion protein [Measles virus strain Moraten] AAF85672.1 fasionprotein fusion protein [Measles virus] AAA56645.1 fusion protein fusionprotein [Measles virus strain Rubeovax] AAF85688.1 fusion protein fusionprotein [Measles virus] AAF85680.1 fusion protein fusion protein[Measles virus] AEF30359.1 fusion protein fusion protein [Measles virus]BAA09957.1 fusion protein fusion protein [Measles virus] AAV84957.1fusion protein fusion protein [Measles virus MeV-eGFP_Edm-tag]AII16636.1 fusion protein fusion protein [Measles virus] ABY58018.1fusion protein fusion protein [Measles virus] BAA19838.1 fusion proteinfusion protein [Measles virus] AAA56641.1 fusion protein F protein[Measles virus] ABK40529.1 fusion protein fusion protein [Measles virus]AAA56652.1 fusion protein fusion protein [Measles virus] ABY58017.1fusion protein fusion protein [Measles virus] ABB71645.1 fusion proteinfusion protein [Measles virus] NP_056922.1 fusion protein fusion protein[Measles virus strain AIK-C] AAF85664.1 fusion protein fusion protein[Measles virus] BAB60865.1 fusion protein fusion protein [Measles virus]BAA09950.1 fusion protein fusion protein [Measles virus strainAEP40403.1 MVi/New York.USA/26.09/3] fusion protein fusion protein[Measles virus] AAA74934.1 fusion protein fusion protein [Measles virus]CAB38075.1 fusion protein fusion protein [Measles virus strainAEP40443.1 MVi/Texas.USA/4.07] fusion protein fusion protein [Measlesvirus] AAF02695.1 fusion protein fusion protein [Measles virus]AAF02696.1 fusion protein fusion protein [Measles virus] AAT99301.1fusion protein fusion protein [Measles virus] ABB71661.1 fusion proteinfusion protein [Measles virus] BAK08874.1 fusion protein fusion protein[Measles virus] AAF02697.1 fusion protein fusion protein [Measles virusgenotype D4] AFY12704.1 fusion protein fusion protein [Measles virusstrain AEP40467.1 MVi/California.USA/16.03] fusion protein fusionprotein [Measles virus genotype D8] AHN07989.1 fusion protein fusionprotein [Measles virus] AAA46421.1 fusion protein fusion protein[Measles virus] AAA56638.1 fusion protein fusion protein [Measles virusstrain AEP40419.1 MVi/Virginia.USA/15.09] fusion protein fusion protein[Measles virus genotype D8] ALE27200.1 fusion protein fusion protein[Measles virus genotype D8] AFY12695.1 fusion protein fusion protein[Measles virus genotype D8] ALE27248.1 fusion protein fusion protein[Measles virus genotype D8] ALE27224.1 fusion protein fusion protein[Measles virus] AAT99300.1 fusion protein fusion protein [Measles virus]BAH96592.1 fusion protein fusion protein [Measles virus strainAEP40459.1 MVi/California.USA/8.04] fusion protein fusion protein[Measles virus genotype D8] AIG94081.1 fusion protein fusion protein[Measles virus] BAA09951.1 fusion protein fusion protein [Measles virusgenotype D8] ALE27194.1 fusion protein fusion protein [Measles virus]BAA33871.1 fusion protein fusion protein [Measles virus strainAEP40427.1 MVi/Washington.USA/18.08/1] fusion protein fusion protein[Measles virus] ABY21182.1 fusion protein fusion protein [Measles virusgenotype D8] ALE27284.1 fusion protein fusion protein [Measles virus]ACA09725.1 fusion protein fusion protein [Measles virus genotype D8]ALE27314.1 fusion protein fusion protein [Measles virus genotype G3]AFY12712.1 fusion protein fusion protein [Measles virus genotype D8]ALE27368.1 fusion protein RecName: Full = Fusion glycoprotein F0;Contains: P35973.1 RecName: Full = Fusion glycoprotein F2; Contains:RecName: Full = Fusion glycoprotein F1; Flags: Precursor fusion proteinfusion protein [Measles virus genotype H1] AIG53713.1 unnamed proteinproduct [Measles virus] CAA34588.1 fusion protein fusion protein[Measles virus] CAA76888.1 fusion protein fusion protein [Measles virusgenotype B3.1] AIY55563.1 fusion protein fusion protein [Measles virus]ADO17330.1 fusion protein fusion protein [Measles virus genotype H1]AIG53703.1 fusion protein fusion protein [Measles virus genotype B3]AGA17208.1 fusion protein fusion protein [Measles virus] AAL29688.1fusion protein fusion protein [Measles virus genotype H1] AIG53706.1fusion protein fusion protein [Measles virus genotype H1] AIG53701.1fusion protein fusion protein [Measles virus genotype B3] ALE27092.1fusion protein fusion protein [Measles virus genotype H1] AIG53714.1fusion protein fusion protein [Measles virus genotype H1] AIG53694.1fusion protein fusion protein [Measles virus genotype H1] AIG53668.1fusion protein fusion protein [Measles virus] ACC86094.1 fusion proteinfusion protein [Measles virus genotype H1] AIG53670.1 fusion proteinfusion protein [Measles virus genotype H1] AIG53707.1 fusion proteinfusion protein [Measles virus genotype B3] AGA17216.1 fusion proteinfusion protein [Measles virus genotype H1] AIG53671.1 fusion proteinfusion protein [Measles virus strain AEP40451.1 MVi/NewJersey.USA/45.05] fusion protein fusion protein [Measles virus genotypeH1] AIG53684.1 fusion protein fusion protein [Measles virus genotype H1]AIG53688.1 fusion protein fusion protein [Measles virus genotype B3]AGA17214.1 fusion protein fusion protein [Measles virus genotype H1]AIG53683.1 fusion protein fusion protein [Measles virus genotype H1]AIG53667.1 fusion protein fusion protein [Measles virus genotype H1]AIG53686.1 fusion protein fusion protein [Measles virus genotype H1]AIG53685.1 fusion protein fusion protein [Measles virus genotype H1]AIG53681.1 unnamed protein product [Measles virus] CAA34589.1 fusionprotein fusion protein [Measles virus genotype H1] AIG53678.1 fusionprotein fusion protein [Measles virus genotype H1] AIG53710.1 fusionprotein fusion protein [Measles virus genotype H1] AIG53669.1 fusionprotein fusion protein [Measles virus genotype H1] AIG53664.1 fusionprotein fusion protein [Measles virus] AAA50547.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53679.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53709.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53672.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53697.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53689.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53676.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53675.1 fusion protein fusionprotein [Measles virus genotype H1] AIG53663.1 fusion protein fusionprotein [Measles virus] BAA19841.1 fusion protein fusion protein[Measles virus] AAF02701.1 fusion protein fusion protein [Measles virusgenotype H1] AIG53680.1 fusion protein fusion protein [Measles virusgenotype H1] AIG53674.1 C protein C protein [Measles virus strainMoraten] AAF85670.1 C protein RecName: Full = Protein C P03424.1 Cprotein C protein [Measles virus] ACN54404.1 C protein C protein[Measles virus] ACN54412.1 C protein RecName: Full = Protein C P35977.1C protein C protein [Measles virus] AAF85678.1 C protein C protein[Measles virus] ABD33998.1 C protein unnamed protein product [Measlesvirus] CAA34586.1 C protein C protein [Measles virus] BAJ51786.1 Cprotein C protein [Measles virus] BAA33869.1 C protein virulence factor[Measles virus] ABO69700.1 C protein C protein [Measles virus]NP_056920.1 C protein C protein [Measles virus] ADO17333.1 C protein Cprotein [Measles virus] ACC86082.1 C protein C protein [Measles virus]BAA33875.1 C protein C protein [Measles virus] ABY21189.1 C protein Cprotein [Measles virus] BAE98296.1 C protein C protein [Measles virus]ADU17782.1 C protein C protein [Measles virus strain AEP40417.1MVi/Virginia.USA/15.09] C protein C protein [Measles virus] ADU17814.1 Cprotein C protein [Measles virus] ADU17798.1 C protein C protein[Measles virus genotype D4] AFY12700.1 C protein C protein [Measlesvirus] ADU17784.1 C protein C protein [Measles virus strain AEP40465.1MVi/California.USA/16.03] C protein C protein [Measles virus] ABB71643.1C protein C protein [Measles virus] AEI91027.1 C protein C protein[Measles virus] ADU17874.1 C protein C protein [Measles virus]ADU17903.1 C protein C protein [Measles virus] CAA34579.1 C protein Cprotein [Measles virus] ADU17790.1 C protein C protein [Measles virus]ADU17800.1 C protein C protein [Measles virus] ABB71667.1 C proteinunnamed protein product [Measles virus] CAA34572.1 C protein C protein[Measles virus strain AEP40433.1 MVi/Arizona.USA/11.08/2] C protein Cprotein [Measles virus] ADU17830.1 C protein C protein [Measles virus]ADU17947.1 C protein C protein [Measles virus] ADU17818.1 C protein Cprotein [Measles virus strain AEP40449.1 MVi/New Jersey.USA/45.05] Cprotein C protein [Measles virus strain AEP40441.1 MVi/Texas.USA/4.07] Cprotein C protein [Measles virus] ADU17864.1 C protein C protein[Measles virus] ADU17838.1 C protein C protein [Measles virus]ADU17881.1 C protein C protein [Measles virus strain AEP40425.1MVi/Washington.USA/18.08/1] C protein C protein [Measles virus]ADU17927.1 C protein C protein [Measles virus] ADU17953.1 C protein Cprotein [Measles virus] ADU17889.1 C protein C protein [Measles virus]ADU17963.1 C protein C protein [Measles virus] ADU17893.1 C protein Cprotein [Measles virus] ADU17820.1 C protein C protein [Measles virus]ABB71651.1 C protein C protein [Measles virus] ADU17786.1 C protein Cprotein [Measles virus] ADU17862.1 C protein C protein [Measles virus]ADU17923.1 C protein C protein [Measles virus] ADU17959.1 C protein Cprotein [Measles virus] ADU17951.1 C protein C protein [Measles virus]ADU17916.1 C protein C protein [Measles virus] ADU17957.1 C protein Cprotein [Measles virus] ADU17925.1 C protein C protein [Measles virus]ADU17901.1 C protein C protein [Measles virus] ADU17887.1 C protein Cprotein [Measles virus] ADU17832.1 C protein C protein [Measles virus]ADU17891.1 C protein C protein [Measles virus] ADU17961.1 C protein Cprotein [Measles virus] ADU17872.1 C protein C protein [Measles virus]ADU17929.1 C protein C protein [Measles virus] ADU17908.1 C protein Cprotein [Measles virus] ADU17910.1 C protein C protein [Measles virus]ADU17921.1 C protein C protein [Measles virus] ADU17824.1 C protein Cprotein [Measles virus strain AEP40473.1 MVi/Pennsylvania.USA/20.09] Cprotein C protein [Measles virus] ADU17828.1 C protein C protein[Measles virus] ADU17812.1 C protein C protein [Measles virus genotypeD8] AFY12692.1 C protein nonstructural C protein [Measles virus]ABA59559.1 C protein RecName: Full = Protein C Q00794.1 C proteinnonstructural C protein [Measles virus] ADO17934.1 C proteinnonstructural C protein [Measles virus] ACJ66773.1 C protein C protein[Measles virus genotype G3] AFY12708.1 C protein RecName: Full = ProteinC P26035.1 C protein C protein [Measles virus] BAA84128.1 nucleoproteinRecName: Full = Nucleoprotein; AltName: Q77M43.1 Full = Nucleocapsidprotein; Short = NP; Short = Protein N nucleoprotein nucleocapsidprotein [Measles virus strain Rubeovax] AAF85683.1 nucleoproteinRecName: Full = Nucleoprotein; AltName: Q89933.1 Full = Nucleocapsidprotein; Short = NP; Short = Protein N nucleoprotein nucleocapsidprotein [Measles virus strain AIK-C] AAF85659.1 nucleoproteinnucleoprotein [Measles virus] ABI54102.1 nucleoprotein nucleoprotein[Measles virus] AAA56643.1 nucleoprotein nucleoprotein [Measles virus]AAC03050.1 nucleoprotein nucleoprotein [Measles virus] AAA18990.1nucleoprotein nucleoprotein [Measles virus] AAA56640.1 nucleoproteinRecName: Full = Nucleoprotein; AltName: P35972.1 Full = Nucleocapsidprotein; Short = NP; Short = Protein N nucleoprotein RecName:Full=Nucleoprotein; AltName: P10050.1 Full = Nucleocapsid protein; Short= NP; Short = Protein N nucleoprotein N protein [Measles virus]BAB60956.1 nucleoprotein RecName: Full = Nucleoprotein; AltName:B1AAA7.1 Full = Nucleocapsid protein; Short = NP; Short = Protein Nnucleoprotein nucleoprotein [Measles virus] AAA18991.1 nucleoproteinnucleoprotein [Measles virus] CAB46894.1 nucleoprotein nucleoprotein[Measles virus] CAB46871.1 nucleoprotein nucleoprotein [Measles virus]CAB46872.1 nucleoprotein nucleoprotein [Measles virus] ABU49606.1nucleoprotein nucleocapsid protein [Measles virus] AAA75494.1nucleoprotein nucleoprotein [Measles virus] CAB46883.1 nucleoproteinnucleoprotein [Measles virus] CAB46892.1 nucleoprotein unnamed proteinproduct [Measles virus] CAA34584.1 nucleoprotein nucleoprotein [Measlesvirus] AAA18997.1 nucleoprotein nucleoprotein [Measles virus] CAB46863.1nucleoprotein nucleoprotein [Measles virus] AEF30352.1 nucleoproteinnucleoprotein [Measles virus] ABI54103.1 nucleoprotein nucleocapsidprotein [Measles virus] AAA46433.1 nucleoprotein nucleoprotein [Measlesvirus] CAB46902.1 nucleoprotein nucleoprotein [Measles virus] CAB46873.1nucleoprotein nucleoprotein [Measles virus] CAB46906.1 nucleoproteinnucleoprotein [Measles virus] AAA74547.1 nucleoprotein nucleoprotein[Measles virus] AAA74537.1 nucleoprotein nucleoprotein [Measles virus]CAB46862.1 nucleoprotein nucleocapsid protein [Measles virus] BAA09961.1nucleoprotein nucleoprotein [Measles virus] AAO15875.1 nucleoproteinnucleoprotein [Measles virus] AAO15871.1 nucleoprotein nucleoprotein[Measles virus] CAB46882.1 nucleoprotein nucleoprotein [Measles virus]CAB60124.1 nucleoprotein nucleoprotein [Measles virus] ABI54104.1nucleoprotein nucleoprotein [Measles virus] CAB46869.1 nucleoproteinnucleoprotein [Measles virus] CAB46880.1 nucleoprotein nucleoprotein[Measles virus] AAA74541.1 nucleoprotein nucleocapsid protein [Measlesvirus strain AEP40446.1 MVi/New Jersey.USA/45.05] nucleoproteinnucleoprotein [Measles virus] ABI54110.1 nucleoprotein nucleoprotein[Measles virus] CAB46903.1 nucleoprotein nucleoprotein [Measles virus]CAB46899.1 nucleoprotein nucleoprotein [Measles virus] CAB46901.1nucleoprotein nucleocapsid protein [Measles virus] ABB71640.1nucleoprotein nucleoprotein [Measles virus] CAB60113.1 nucleoproteinnucleoprotein [Measles virus] CAB60114.1 nucleoprotein nucleoprotein[Measles virus] CAB60116.1 nucleoprotein nucleoprotein [Measles virus]CAB46895.1 nucleoprotein nucleoprotein [Measles virus] CAB60121.1nucleoprotein nucleoprotein [Measles virus] ABI54111.1 nucleoproteinnucleoprotein [Measles virus] CAB46889.1 nucleoprotein nucleoprotein[Measles virus] CAB46898.1 nucleoprotein nucleoprotein [Measles virusgenotype B3] ALE27083.1 nucleoprotein nucleoprotein [Measles virus]CAB60118.1 nucleoprotein nucleocapsid protein [Measles virus] CAA34570.1nucleoprotein nucleoprotein [Measles virus] AAC29443.1 nucleoproteinnucleocapsid protein [Measles virus strain AEP40422.1MVi/Washington.USA/18.08/1] nucleoprotein nucleoprotein [Measles virus]AAO15872.1 nucleoprotein nucleoprotein [Measles virus] CAB46874.1nucleoprotein nucleoprotein [Measles virus] AAA74550.1 nucleoproteinnucleocapsid protein [Measles virus] ABB71648.1 nucleoproteinnucleoprotein [Measles virus] CAB46900.1 nucleoprotein nucleoprotein[Measles virus] BAH22440.1 nucleoprotein nucleocapsid protein [Measlesvirus] AAA46432.1 nucleoprotein nucleocapsid protein [Measles virus]BAA33867.1 nucleoprotein nucleoprotein [Measles virus] AAA74539.1nucleoprotein nucleoprotein [Measles virus] CAB60115.1 nucleoproteinnucleoprotein [Measles virus] CAB60123.1 nucleoprotein nucleocapsidprotein [Measles virus] ABB71664.1 nucleoprotein nucleoprotein [Measlesvirus] CAB60125.1 nucleoprotein nucleoprotein [Measles virus] AAA74546.1nucleoprotein nucleoprotein [Measles virus] CAB46886.1 nucleoproteinnucleoprotein [Measles virus] BAH22350.1 nucleoprotein nucleoprotein[Measles virus] CAB46867.1 nucleoprotein nucleocapsid protein [Measlesvirus] BAA09954.1 nucleoprotein nucleoprotein [Measles virus] AAO15873.1nucleoprotein nucleocapsid protein [Measles virus] AEP95735.1nucleoprotein nucleoprotein [Measles virus] AAL37726.1 nucleoproteinnucleoprotein [Measles virus] AAA74549.1 nucleoprotein RecName: Full =Nucleoprotein; AltName: P26030.1 Full = Nucleocapsid protein; Short =NP; Short = Protein N nucleoprotein nucleoprotein [Measles virusETH55/99] AAK07777.1 nucleoprotein nucleoprotein [Measles virus genotypeB3] AGA17238.1 nucleoprotein nucleoprotein [Measles virus] AEF30351.1nucleoprotein nucleoprotein [Measles virus genotype B3] AGA17242.1nucleoprotein nucleoprotein [Measles virus ETH54/98] AAK07776.1nucleoprotein nucleoprotein [Measles virus] AAA74548.1 nucleoproteinnucleoprotein [Measles virus] AAA19221.1 nucleoprotein nucleoprotein[Measles virus] AAC03039.1 nucleoprotein nucleoprotein [Measles virus]AAA19223.1 nucleoprotein nucleoprotein [Measles virus genotype B3]AGA17241.1 nucleoprotein nucleoprotein [Measles virus] CAB60122.1nucleoprotein nucleoprotein [Measles virus] CAC34599.1 nucleoproteinnucleoprotein [Measles virus] AAC03042.1 nucleoprotein nucleoprotein[Measles virus] CAC34604.1 nucleoprotein nucleoprotein [Measles virus]AAA74544.1 nucleoprotein nucleocapsid protein [Measles virus]NP_056918.1 V Protein RecName: Full = Non-structural protein V Q9IC37.1V Protein RecName: Full = Non-structural protein V Q9EMA9.1 V Protein Vprotein [Measles virus] ACN54411.1 V Protein V protein [Measles virus]ACN54403.1 V Protein V protein [Measles virus] AEP95742.1 V Protein Vprotein [Measles virus strain AEP40416.1 MVi/Virginia.USA/15.09] VProtein V protein [Measles virus] ADU17801.1 V Protein V protein[Measles virus] ADU17849.1 V Protein V protein [Measles virus]ABB71642.1 V Protein V protein [Measles virus genotype D8] AFY12693.1 VProtein V protein [Measles virus] YP_003873249.2 V Protein V protein[Measles virus strain AEP40432.1 MVi/Arizona.USA/11.08/2] V ProteinRecName: Full = Non-structural protein V P26036.1 V Protein V protein[Measles virus strain AEP40464.1 MVi/California.USA/16.03] V Protein Vprotein [Measles virus strain AEP40456.1 MVi/California.USA/8.04] VProtein V protein [Measles virus] ABY21188.1 V Protein V protein[Measles virus strain AEP40424.1 MVi/Washington.USA/18.08/1] V Protein Vprotein [Measles virus] BAH96581.1 V Protein V protein [Measles virus]ABB71666.1 V Protein RecName: Full = Non-structural protein V P60168.1 VProtein V protein [Measles virus] BAH96589.1 V Protein V protein[Measles virus] ADU17954.1 V Protein V protein [Measles virus strainAEP40400.1 MVi/New York.USA/26.09/3] V Protein V protein [Measles virus]ABY21196.1 V Protein virulence factor [Measles virus] ABO69701.1 VProtein V protein [Measles virus] ABB71650.1 V Protein V protein[Measles virus] ACC86086.1 V Protein V protein [Measles virus genotypeD4] AFY12702.1 V Protein V protein [Measles virus strain AEP40448.1MVi/New Jersey.USA/45.05] V Protein V protein [Measles virus] BAE98295.1V Protein V protein [Measles virus] ACC86083.1 V Protein V protein[Measles virus] ACU5139.1 V Protein V protein [Measles virus] ADO17334.1V Protein V protein [Measles virus] ADU17930.1 V Protein V protein[Measles virus genotype G3] AFY12710.1 V Protein V protein [Measlesvirus strain AEP40472.1 MVi/Pennsylvania.USA/20.09] V Proteinphosphoprotein [Measles virus] ADU17839.1 V Protein V protein [Measlesvirus] ADU17894.1 V Protein V protein [Measles virus] ACN50010.1 VProtein V protein [Measles virus] ADU17892.1 unnamed protein product[Measles virus] CAA34585.1 V Protein V protein [Measles virus]ABD33997.1

TABLE 16 SEQ ID Name Sequence NO: Flagellin Nucleic Acid SequencesNT (5′ TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTAT 51 UTR, ORF,AGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG 3′ UTR)AGCCACCATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACTGCTATCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCGAATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGATGCCTACACCCCGAAAGAAACTGCTGTAACCGTTGATAAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCAGAGCAATACTGATATCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATGGTCAATACTATTTAGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGATACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAACCCACAACCAAATTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGGTTACTGGCGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGTGGCTATGCAGTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAACCACTACTTATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAGTTGTTACTGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTTGACAAACATAACTTCAGAACAGGCGGTGAGCTTAAAGAGGTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTGACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGCCCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGG C ORFATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCC 52 Sequence,AGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACTGCTAT NTCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCGAATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGATGCCTACACCCCGAAAGAAACTGCTGTAACCGTTGATAAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCAGAGCAATACTGATATCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATGGTCAATACTATTTAGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGATACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAACCCACAACCAAATTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGGTTACTGGCGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGTGGCTATGCAGTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAACCACTACTTATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAGTTGTTACTGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTTGACAAACATAACTTCAGAACAGGCGGTGAGCTTAAAGAGGTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTGACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGCCCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTT TACTGCGT mRNAG*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA 53 SequenceGAGCCACCAUGGCACAAGUCAUUAAUACAAACAGCCUGUCGC (assumesUGUUGACCCAGAAUAACCUGAACAAAUCCCAGUCCGCACUGG T100 tail)GCACUGCUAUCGAGCGUUUGUCUUCCGGUCUGCGUAUCAACAGCGCGAAAGACGAUGCGGCAGGACAGGCGAUUGCUAACCGUUUUACCGCGAACAUCAAAGGUCUGACUCAGGCUUCCCGUAACGCUAACGACGGUAUCUCCAUUGCGCAGACCACUGAAGGCGCGCUGAACGAAAUCAACAACAACCUGCAGCGUGUGCGUGAACUGGCGGUUCAGUCUGCGAAUGGUACUAACUCCCAGUCUGACCUCGACUCCAUCCAGGCUGAAAUCACCCAGCGCCUGAACGAAAUCGACCGUGUAUCCGGCCAGACUCAGUUCAACGGCGUGAAAGUCCUGGCGCAGGACAACACCCUGACCAUCCAGGUUGGUGCCAACGACGGUGAAACUAUCGAUAUUGAUUUAAAAGAAAUCAGCUCUAAAACACUGGGACUUGAUAAGCUUAAUGUCCAAGAUGCCUACACCCCGAAAGAAACUGCUGUAACCGUUGAUAAAACUACCUAUAAAAAUGGUACAGAUCCUAUUACAGCCCAGAGCAAUACUGAUAUCCAAACUGCAAUUGGCGGUGGUGCAACGGGGGUUACUGGGGCUGAUAUCAAAUUUAAAGAUGGUCAAUACUAUUUAGAUGUUAAAGGCGGUGCUUCUGCUGGUGUUUAUAAAGCCACUUAUGAUGAAACUACAAAGAAAGUUAAUAUUGAUACGACUGAUAAAACUCCGUUGGCAACUGCGGAAGCUACAGCUAUUCGGGGAACGGCCACUAUAACCCACAACCAAAUUGCUGAAGUAACAAAAGAGGGUGUUGAUACGACCACAGUUGCGGCUCAACUUGCUGCAGCAGGGGUUACUGGCGCCGAUAAGGACAAUACUAGCCUUGUAAAACUAUCGUUUGAGGAUAAAAACGGUAAGGUUAUUGAUGGUGGCUAUGCAGUGAAAAUGGGCGACGAUUUCUAUGCCGCUACAUAUGAUGAGAAAACAGGUGCAAUUACUGCUAAAACCACUACUUAUACAGAUGGUACUGGCGUUGCUCAAACUGGAGCUGUGAAAUUUGGUGGCGCAAAUGGUAAAUCUGAAGUUGUUACUGCUACCGAUGGUAAGACUUACUUAGCAAGCGACCUUGACAAACAUAACUUCAGAACAGGCGGUGAGCUUAAAGAGGUUAAUACAGAUAAGACUGAAAACCCACUGCAGAAAAUUGAUGCUGCCUUGGCACAGGUUGAUACACUUCGUUCUGACCUGGGUGCGGUUCAGAACCGUUUCAACUCCGCUAUCACCAACCUGGGCAAUACCGUAAAUAACCUGUCUUCUGCCCGUAGCCGUAUCGAAGAUUCCGACUACGCAACCGAAGUCUCCAACAUGUCUCGCGCGCAGAUUCUGCAGCAGGCCGGUACCUCCGUUCUGGCGCAGGCGAACCAGGUUCCGCAAAACGUCCUCUCUUUACUGCGUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG Flagellin mRNA SequencesNT (5′ UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACU 81 UTR, ORF,AUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 3′ UTR)AGAGCCACCAUGGCACAAGUCAUUAAUACAAACAGCCUGUCGCUGUUGACCCAGAAUAACCUGAACAAAUCCCAGUCCGCACUGGGCACUGCUAUCGAGCGUUUGUCUUCCGGUCUGCGUAUCAACAGCGCGAAAGACGAUGCGGCAGGACAGGCGAUUGCUAACCGUUUUACCGCGAACAUCAAAGGUCUGACUCAGGCUUCCCGUAACGCUAACGACGGUAUCUCCAUUGCGCAGACCACUGAAGGCGCGCUGAACGAAAUCAACAACAACCUGCAGCGUGUGCGUGAACUGGCGGUUCAGUCUGCGAAUGGUACUAACUCCCAGUCUGACCUCGACUCCAUCCAGGCUGAAAUCACCCAGCGCCUGAACGAAAUCGACCGUGUAUCCGGCCAGACUCAGUUCAACGGCGUGAAAGUCCUGGCGCAGGACAACACCCUGACCAUCCAGGUUGGUGCCAACGACGGUGAAACUAUCGAUAUUGAUUUAAAAGAAAUCAGCUCUAAAACACUGGGACUUGAUAAGCUUAAUGUCCAAGAUGCCUACACCCCGAAAGAAACUGCUGUAACCGUUGAUAAAACUACCUAUAAAAAUGGUACAGAUCCUAUUACAGCCCAGAGCAAUACUGAUAUCCAAACUGCAAUUGGCGGUGGUGCAACGGGGGUUACUGGGGCUGAUAUCAAAUUUAAAGAUGGUCAAUACUAUUUAGAUGUUAAAGGCGGUGCUUCUGCUGGUGUUUAUAAAGCCACUUAUGAUGAAACUACAAAGAAAGUUAAUAUUGAUACGACUGAUAAAACUCCGUUGGCAACUGCGGAAGCUACAGCUAUUCGGGGAACGGCCACUAUAACCCACAACCAAAUUGCUGAAGUAACAAAAGAGGGUGUUGAUACGACCACAGUUGCGGCUCAACUUGCUGCAGCAGGGGUUACUGGCGCCGAUAAGGACAAUACUAGCCUUGUAAAACUAUCGUUUGAGGAUAAAAACGGUAAGGUUAUUGAUGGUGGCUAUGCAGUGAAAAUGGGCGACGAUUUCUAUGCCGCUACAUAUGAUGAGAAAACAGGUGCAAUUACUGCUAAAACCACUACUUAUACAGAUGGUACUGGCGUUGCUCAAACUGGAGCUGUGAAAUUUGGUGGCGCAAAUGGUAAAUCUGAAGUUGUUACUGCUACCGAUGGUAAGACUUACUUAGCAAGCGACCUUGACAAACAUAACUUCAGAACAGGCGGUGAGCUUAAAGAGGUUAAUACAGAUAAGACUGAAAACCCACUGCAGAAAAUUGAUGCUGCCUUGGCACAGGUUGAUACACUUCGUUCUGACCUGGGUGCGGUUCAGAACCGUUUCAACUCCGCUAUCACCAACCUGGGCAAUACCGUAAAUAACCUGUCUUCUGCCCGUAGCCGUAUCGAAGAUUCCGACUACGCAACCGAAGUCUCCAACAUGUCUCGCGCGCAGAUUCUGCAGCAGGCCGGUACCUCCGUUCUGGCGCAGGCGAACCAGGUUCCGCAAAACGUCCUCUCUUUACUGCGUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC ORFAUGGCACAAGUCAUUAAUACAAACAGCCUGUCGCUGUUGACC 82 Sequence,CAGAAUAACCUGAACAAAUCCCAGUCCGCACUGGGCACUGCU NTAUCGAGCGUUUGUCUUCCGGUCUGCGUAUCAACAGCGCGAAAGACGAUGCGGCAGGACAGGCGAUUGCUAACCGUUUUACCGCGAACAUCAAAGGUCUGACUCAGGCUUCCCGUAACGCUAACGACGGUAUCUCCAUUGCGCAGACCACUGAAGGCGCGCUGAACGAAAUCAACAACAACCUGCAGCGUGUGCGUGAACUGGCGGUUCAGUCUGCGAAUGGUACUAACUCCCAGUCUGACCUCGACUCCAUCCAGGCUGAAAUCACCCAGCGCCUGAACGAAAUCGACCGUGUAUCCGGCCAGACUCAGUUCAACGGCGUGAAAGUCCUGGCGCAGGACAACACCCUGACCAUCCAGGUUGGUGCCAACGACGGUGAAACUAUCGAUAUUGAUUUAAAAGAAAUCAGCUCUAAAACACUGGGACUUGAUAAGCUUAAUGUCCAAGAUGCCUACACCCCGAAAGAAACUGCUGUAACCGUUGAUAAAACUACCUAUAAAAAUGGUACAGAUCCUAUUACAGCCCAGAGCAAUACUGAUAUCCAAACUGCAAUUGGCGGUGGUGCAACGGGGGUUACUGGGGCUGAUAUCAAAUUUAAAGAUGGUCAAUACUAUUUAGAUGUUAAAGGCGGUGCUUCUGCUGGUGUUUAUAAAGCCACUUAUGAUGAAACUACAAAGAAAGUUAAUAUUGAUACGACUGAUAAAACUCCGUUGGCAACUGCGGAAGCUACAGCUAUUCGGGGAACGGCCACUAUAACCCACAACCAAAUUGCUGAAGUAACAAAAGAGGGUGUUGAUACGACCACAGUUGCGGCUCAACUUGCUGCAGCAGGGGUUACUGGCGCCGAUAAGGACAAUACUAGCCUUGUAAAACUAUCGUUUGAGGAUAAAAACGGUAAGGUUAUUGAUGGUGGCUAUGCAGUGAAAAUGGGCGACGAUUUCUAUGCCGCUACAUAUGAUGAGAAAACAGGUGCAAUUACUGCUAAAACCACUACUUAUACAGAUGGUACUGGCGUUGCUCAAACUGGAGCUGUGAAAUUUGGUGGCGCAAAUGGUAAAUCUGAAGUUGUUACUGCUACCGAUGGUAAGACUUACUUAGCAAGCGACCUUGACAAACAUAACUUCAGAACAGGCGGUGAGCUUAAAGAGGUUAAUACAGAUAAGACUGAAAACCCACUGCAGAAAAUUGAUGCUGCCUUGGCACAGGUUGAUACACUUCGUUCUGACCUGGGUGCGGUUCAGAACCGUUUCAACUCCGCUAUCACCAACCUGGGCAAUACCGUAAAUAACCUGUCUUCUGCCCGUAGCCGUAUCGAAGAUUCCGACUACGCAACCGAAGUCUCCAACAUGUCUCGCGCGCAGAUUCUGCAGCAGGCCGGUACCUCCGUUCUGGCGCAGGCGAACCAGGUUCCGCAAAAC GUCCUCUCUUUACUGCGU mRNAG*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA 83 SequenceGAGCCACCAUGGCACAAGUCAUUAAUACAAACAGCCUGUCGC (assumesUGUUGACCCAGAAUAACCUGAACAAAUCCCAGUCCGCACUGG T100 tail)GCACUGCUAUCGAGCGUUUGUCUUCCGGUCUGCGUAUCAACAGCGCGAAAGACGAUGCGGCAGGACAGGCGAUUGCUAACCGUUUUACCGCGAACAUCAAAGGUCUGACUCAGGCUUCCCGUAACGCUAACGACGGUAUCUCCAUUGCGCAGACCACUGAAGGCGCGCUGAACGAAAUCAACAACAACCUGCAGCGUGUGCGUGAACUGGCGGUUCAGUCUGCGAAUGGUACUAACUCCCAGUCUGACCUCGACUCCAUCCAGGCUGAAAUCACCCAGCGCCUGAACGAAAUCGACCGUGUAUCCGGCCAGACUCAGUUCAACGGCGUGAAAGUCCUGGCGCAGGACAACACCCUGACCAUCCAGGUUGGUGCCAACGACGGUGAAACUAUCGAUAUUGAUUUAAAAGAAAUCAGCUCUAAAACACUGGGACUUGAUAAGCUUAAUGUCCAAGAUGCCUACACCCCGAAAGAAACUGCUGUAACCGUUGAUAAAACUACCUAUAAAAAUGGUACAGAUCCUAUUACAGCCCAGAGCAAUACUGAUAUCCAAACUGCAAUUGGCGGUGGUGCAACGGGGGUUACUGGGGCUGAUAUCAAAUUUAAAGAUGGUCAAUACUAUUUAGAUGUUAAAGGCGGUGCUUCUGCUGGUGUUUAUAAAGCCACUUAUGAUGAAACUACAAAGAAAGUUAAUAUUGAUACGACUGAUAAAACUCCGUUGGCAACUGCGGAAGCUACAGCUAUUCGGGGAACGGCCACUAUAACCCACAACCAAAUUGCUGAAGUAACAAAAGAGGGUGUUGAUACGACCACAGUUGCGGCUCAACUUGCUGCAGCAGGGGUUACUGGCGCCGAUAAGGACAAUACUAGCCUUGUAAAACUAUCGUUUGAGGAUAAAAACGGUAAGGUUAUUGAUGGUGGCUAUGCAGUGAAAAUGGGCGACGAUUUCUAUGCCGCUACAUAUGAUGAGAAAACAGGUGCAAUUACUGCUAAAACCACUACUUAUACAGAUGGUACUGGCGUUGCUCAAACUGGAGCUGUGAAAUUUGGUGGCGCAAAUGGUAAAUCUGAAGUUGUUACUGCUACCGAUGGUAAGACUUACUUAGCAAGCGACCUUGACAAACAUAACUUCAGAACAGGCGGUGAGCUUAAAGAGGUUAAUACAGAUAAGACUGAAAACCCACUGCAGAAAAUUGAUGCUGCCUUGGCACAGGUUGAUACACUUCGUUCUGACCUGGGUGCGGUUCAGAACCGUUUCAACUCCGCUAUCACCAACCUGGGCAAUACCGUAAAUAACCUGUCUUCUGCCCGUAGCCGUAUCGAAGAUUCCGACUACGCAACCGAAGUCUCCAACAUGUCUCGCGCGCAGAUUCUGCAGCAGGCCGGUACCUCCGUUCUGGCGCAGGCGAACCAGGUUCCGCAAAACGUCCUCUCUUUACUGCGUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG

TABLE 17 Flagellin Amino Acid Sequences SEQ ID Name Sequence NO: ORFMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA 54 Sequence,GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV AARELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKEISSKTLGLDKLNVQDAYTPKETAVTVDKTTYKNGTDPITAQSNTDIQTAIGGGATGVTGADIKFKDGQYYLDVKGGASAGVYKATYDETTKKVNIDTTDKTPLATAEATAIRGTATITHNQIAEVTKEGVDTTTVAAQLAAAGVTGADKDNTSLVKLSFEDKNGKVIDGGYAVKMGDDFYAATYDEKTGAITAKTTTYTDGTGVAQTGAVKFGGANGKSEVVTATDGKTYLASDLDKHNFRTGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR Flagellin-MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA 55 GS linker-GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV circumsporRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVL ozoiteAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDT proteinAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKY (CSP)YAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRGGGGSGGGGSMMAPDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEQYLKKIKNSISTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKCS SVFNVVNS Flagellin-MMAPDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANP 56 RPVTNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQG linker-HNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEQYLKKIKNSIS circumsporTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKC ozoite SSVFNVVNSRPVTMAQVINTNSLSLLTQNNLNKSQSALGTAIERLS proteinSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTE (CSP)GALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSL LR

TABLE 18 Human Metapneumovirus Mutant Amino Acid Sequences SEQ ID StrainSequence NO: HMPV_SC_DSCAV1_4MMVMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  85VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAICKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLAFAVRELKDFVSKNLTRALNKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILCGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_DSTRIC_4MMVMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  86VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAICKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILCGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEHQWHVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_DM_Krarup_T74LD185PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  87VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_TM_Krarup_T74LD185PD454NMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  88VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPENQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_4M_Krarup_T74LS170LD185PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  89VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVLKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_5M_Krarup_T74LS170LD185PD454NMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  90VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVLKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPENQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_DM_Krarup_E51PT74LMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLP  91VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_TM_Krarup_E51PT74LD454NMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLP  92VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPENQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_StabilizeAlpha_T74LMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  93VGDVENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_StabilizeAlpha_V55LMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  94VGDLENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_StabilizeAlpha_S170LMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  95VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVLKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_StabilizeAlpha_T174WMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  96VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLWRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_SC_4M_Stabilize-MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  97Alpha_V55LT74LS170LT174WVGDLENLTCSDGPSLIKTELDLLKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVLKNLWRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_ProlineStab_E51PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLP  98VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_ProlineStab_D185PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE  99VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_ProlineStab_D183PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 100VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCPIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_ProlineStab_E131PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 101VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLPSEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_ProlineStab_D447PMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 102VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFPPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_TrimerRepulsionD454NMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 103VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPENQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_TrimerRepulsionE453NMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 104VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN HMPV_StabilizeAlphaF196WMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLE 105VGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPGSGSFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQWNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFQVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHN

TABLE 19 SEQ ID Strain Nucleic Acid Sequence NO:Human Metapneumovirus Mutant Nucleic Acid Sequences HMPV_SC_DSCAV1_4MMVATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 106CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCTGCAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCTTTGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCCTGAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGTGTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCAACGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_SC_DSTRIC_4MMV ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 107CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCTGCAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGTGTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGCACCAGTGGCATGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_SC_DM_Krarup_T74LD185P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 108CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCCCTGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_TM_Krarup_T74LD185PD454NATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 109CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCCCTGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGAACCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_4M_Krarup_T74LS170LD185PATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 110CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGCTTAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCCCTGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_5M_Krarup_T74LS170LD185PD454NATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 111CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGCTTAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCCCTGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGAACCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_DM_Krarup_E51PT74L ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 112CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGCCTGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_TM_Krarup_E51PT74LD454N ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA113 CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGCCTGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGAACCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_StabilizeAlpha_T74L ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 114CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_SC_StabilizeAlpha_V55L ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 115CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACCTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_SC_StabilizeAlpha_S170L ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA116 CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGCTTAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_SC_StabilizeAlpha_T174W ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA117 CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGTGGCGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_SC_4M_Stabilize- ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 118Alpha_V55LT74LS170LT174W CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACCTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGCTCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGCTTAAGAACCTGTGGCGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_ProlineStab_E51P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 119CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGCCTGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_ProlineStab_D185P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 120CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCCCTGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_ProlineStab_D183P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 121CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCCCTATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_ProlineStab_E131P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 122CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGCCTAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTGACCAA CAATGGCTTCATCCCTCACAACHMPV_ProlineStab_D447P ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 123CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCCCACCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_TrimerRepulsionD454N ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 124CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGAACCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_TrimerRepulsionE453N ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 125CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTTCAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTCAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHMPV_StabilizeAlphaF196W ATGAGCTGGAAGGTGGTCATCATCTTCAGCCTGCTGATCA 126CACCTCAGCACGGCCTGAAAGAGAGCTACCTGGAAGAGTCCTGCAGCACCATCACAGAGGGCTACCTGTCTGTGCTGAGAACCGGCTGGTACACCAACGTGTTCACACTGGAAGTGGGCGACGTCGAGAATCTGACATGCTCTGATGGCCCTAGCCTGATCAAGACCGAGCTGGATCTGACCAAGAGCGCCCTGAGAGAACTCAAGACCGTGTCTGCCGATCAGCTGGCCAGAGAGGAACAGATCGAGAATCCTGGCAGCGGCAGCTTTGTGCTGGGAGCCATTGCTCTTGGAGTGGCTGCTGCTGCAGCTGTTACAGCAGGCGTGGCCATCGCTAAGACCATCAGACTGGAAAGCGAAGTGACCGCCATCAACAACGCCCTGAAGAAGACAAACGAGGCCGTCAGCACACTCGGCAATGGCGTTAGAGTGCTGGCCACAGCCGTGCGCGAGCTGAAGGACTTCGTGTCCAAGAACCTGACACGGGCCATTAACAAGAACAAGTGCGACATCGACGACCTGAAGATGGCCGTGTCCTTTAGCCAGTGGAACCGGCGGTTTCTGAACGTCGTGCGGCAGTTTAGCGACAACGCCGGAATCACACCAGCCATCAGCCTGGACCTGATGACAGATGCTGAGCTGGCTAGAGCCGTGCCTAACATGCCTACATCTGCCGGCCAGATCAAGCTGATGCTCGAGAATAGAGCCATGGTCCGACGGAAAGGCTTCGGCATTCTGATTGGCGTGTACGGCAGCAGCGTGATCTATATGGTGCAGCTGCCTATCTTCGGCGTGATCGACACACCCTGCTGGATTGTGAAGGCCGCTCCTAGCTGTAGCGAGAAGAAGGGCAATTACGCCTGCCTGCTGAGAGAGGACCAAGGCTGGTATTGTCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAGGACTGCGAGACAAGAGGCGACCACGTGTTCTGTGATACCGCCGCTGGAATCAATGTGGCCGAGCAGAGCAAAGAGTGCAACATCAACATCAGCACCACCAACTATCCCTGCAAGGTGTCCACCGGCAGGCACCCTATTTCTATGGTGGCTCTGTCTCCTCTGGGAGCCCTGGTGGCTTGTTATAAGGGCGTGTCCTGTAGCATCGGCAGCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGCTGCAGCTACATCACCAACCAGGACGCCGATACCGTGACCATCGACAACACCGTGTATCAGCTGAGCAAGGTGGAAGGCGAACAGCACGTGATCAAGGGCAGACCTGTGTCCAGCAGCTTCGACCCTATCAAGTTCCCTGAGGATCAGTTCCAGGTGGCCCTGGACCAGGTGTTCGAGAACATCGAGAATTCCCAGGCTCTGGTGGACCAGTCCAACAGAATCCTGTCTAGCGCCGAGAAGGGAAACACCGGCTTCATCATCGTGATCATCCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGTCCATCTTCATCATTATCAAGAAGACCAAGAAGCCCACCGGCGCTCCTCCAGAACTGAGCGGAGTG ACCAACAATGGCTTCATCCCTCACAACHuman Metapneumovirus mRNA Sequences HMPV_SC_DSCAV1_4MMVAUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 127CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCUGCAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCUUUGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCCUGAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGUGUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCAACGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_DSURIC_4MMV AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 128CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCUGCAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGUGUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGCACCAGUGGCAUGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_DM_Krarup_U74LD185P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 129CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCCCUGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_UM_Krarup_U74LD185PD454N AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU130 CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCCCUGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGAACCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_4M_Krarup_U74LS170LD185P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU131 CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGCUUAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCCCUGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_5M_Krarup_U74LS170LD185PD454NAUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 132CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGCUUAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCCCUGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGAACCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_DM_Krarup_E51PU74L AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 133CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGCCUGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_UM_Krarup_E51PU74LD454N AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU134 CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGCCUGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGAACCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_SUabilizeAlpha_U74L AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 135CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_SUabilizeAlpha_V55L AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 136CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACCUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_SUabilizeAlpha_S170L AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 137CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGCUUAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_SUabilizeAlpha_U174W AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 138CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGUGGCGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_SC_4M_SUabilize- AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 139Alpha_V55LU74LS170LU174W CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACCUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGCUCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGCUUAAGAACCUGUGGCGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_ProlineSUab_E51P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 140CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGCCUGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_ProlineSUab_D185P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 141CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCCCUGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_ProlineSUab_D183P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 142CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCCCUAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_ProlineSUab_E131P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 143CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGCCUAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_ProlineSUab_D447P AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 144CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCCCACCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAACHMPV_UrimerRepulsionD454N AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 145CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGAACCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_UrimerRepulsionE453N AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 146CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUUCAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUCAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAGU GACCAACAAUGGCUUCAUCCCUCACAACHMPV_SUabilizeAlphaF196W AUGAGCUGGAAGGUGGUCAUCAUCUUCAGCCUGCUGAU 147CACACCUCAGCACGGCCUGAAAGAGAGCUACCUGGAAGAGUCCUGCAGCACCAUCACAGAGGGCUACCUGUCUGUGCUGAGAACCGGCUGGUACACCAACGUGUUCACACUGGAAGUGGGCGACGUCGAGAAUCUGACAUGCUCUGAUGGCCCUAGCCUGAUCAAGACCGAGCUGGAUCUGACCAAGAGCGCCCUGAGAGAACUCAAGACCGUGUCUGCCGAUCAGCUGGCCAGAGAGGAACAGAUCGAGAAUCCUGGCAGCGGCAGCUUUGUGCUGGGAGCCAUUGCUCUUGGAGUGGCUGCUGCUGCAGCUGUUACAGCAGGCGUGGCCAUCGCUAAGACCAUCAGACUGGAAAGCGAAGUGACCGCCAUCAACAACGCCCUGAAGAAGACAAACGAGGCCGUCAGCACACUCGGCAAUGGCGUUAGAGUGCUGGCCACAGCCGUGCGCGAGCUGAAGGACUUCGUGUCCAAGAACCUGACACGGGCCAUUAACAAGAACAAGUGCGACAUCGACGACCUGAAGAUGGCCGUGUCCUUUAGCCAGUGGAACCGGCGGUUUCUGAACGUCGUGCGGCAGUUUAGCGACAACGCCGGAAUCACACCAGCCAUCAGCCUGGACCUGAUGACAGAUGCUGAGCUGGCUAGAGCCGUGCCUAACAUGCCUACAUCUGCCGGCCAGAUCAAGCUGAUGCUCGAGAAUAGAGCCAUGGUCCGACGGAAAGGCUUCGGCAUUCUGAUUGGCGUGUACGGCAGCAGCGUGAUCUAUAUGGUGCAGCUGCCUAUCUUCGGCGUGAUCGACACACCCUGCUGGAUUGUGAAGGCCGCUCCUAGCUGUAGCGAGAAGAAGGGCAAUUACGCCUGCCUGCUGAGAGAGGACCAAGGCUGGUAUUGUCAGAACGCCGGCAGCACCGUGUACUACCCUAACGAGAAGGACUGCGAGACAAGAGGCGACCACGUGUUCUGUGAUACCGCCGCUGGAAUCAAUGUGGCCGAGCAGAGCAAAGAGUGCAACAUCAACAUCAGCACCACCAACUAUCCCUGCAAGGUGUCCACCGGCAGGCACCCUAUUUCUAUGGUGGCUCUGUCUCCUCUGGGAGCCCUGGUGGCUUGUUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAACAGAGUGGGCAUCAUCAAGCAGCUGAACAAGGGCUGCAGCUACAUCACCAACCAGGACGCCGAUACCGUGACCAUCGACAACACCGUGUAUCAGCUGAGCAAGGUGGAAGGCGAACAGCACGUGAUCAAGGGCAGACCUGUGUCCAGCAGCUUCGACCCUAUCAAGUUCCCUGAGGAUCAGUUCCAGGUGGCCCUGGACCAGGUGUUCGAGAACAUCGAGAAUUCCCAGGCUCUGGUGGACCAGUCCAACAGAAUCCUGUCUAGCGCCGAGAAGGGAAACACCGGCUUCAUCAUCGUGAUCAUCCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGUCCAUCUUCAUCAUUAUCAAGAAGACCAAGAAGCCCACCGGCGCUCCUCCAGAACUGAGCGGAG UGACCAACAAUGGCUUCAUCCCUCACAAC

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

What is claimed is:
 1. A method comprising administering to a subject amessenger ribonucleic acid (mRNA) comprising an open reading frameencoding a betacoronavirus (BetaCoV) S protein or S protein subunitformulated in a lipid nanoparticle in an effective amount to induce inthe subject an immune response to the BetaCoV S protein or S proteinsubunit, wherein the lipid nanoparticle comprises 20-60 mol % ionizablecationic lipid, 5-25 mol % neutral lipid, 25-55 mol % cholesterol, and0.5-15 mol % PEG-modified lipid.
 2. The method of claim 1, wherein theopen reading frame encodes a BetaCoV S protein.
 3. The method of claim2, wherein the immune response is a neutralizing antibody responsespecific to the BetaCoV S protein.
 4. The method of claim 1, wherein theopen reading frame encodes a BetaCoV S protein subunit selected from anS1 subunit and an S2 subunit.
 5. The method of claim 4, wherein theimmune response is a neutralizing antibody response specific to theBetaCoV S protein subunit.
 6. The method of claim 1, wherein the mRNAformulated in a lipid nanoparticle is administered intramuscularly. 7.The method of claim 1, wherein the mRNA further comprises a 5′untranslated region and a 3′ untranslated region.
 8. The method of claim1, wherein the mRNA further comprises a poly(A) tail.
 9. The method ofclaim 1, wherein the mRNA further comprises a 5′ cap analog.
 10. Themethod of claim 9, wherein the 5′ cap analog is 7mG(5′)ppp(5′)NlmpNp.11. The method of claim 1, wherein the mRNA comprises a chemicalmodification.
 12. The method of claim 11, wherein the chemicalmodification is a 1-methylpseudouridine modification or a1-ethylpseudouridine modification.
 13. The method of claim 11, whereinat least 80% of the uracil in the open reading frame of the mRNA has achemical modification.
 14. The method of claim 1, wherein the lipidnanoparticle comprises 50 mol % ionizable cationic lipid, 10 mol %neutral lipid, 38.5 mol % cholesterol, and 1.5 mol % PEG-modified lipid.15. The method of claim 1, wherein the ionizable cationic lipid isCompound
 25. 16. The method of claim 1, wherein the neutral lipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and the PEG-modifiedlipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(PEG-DMG).
 17. A method comprising administering to a subject an mRNAcomprising a 5′ cap analog, a 5′ untranslated region, an open readingframe encoding a BetaCoV S protein or S protein subunit, a 3′untranslated region, and a poly(A) tail formulated in a lipidnanoparticle in an effective amount to induce in the subject an immuneresponse to the BetaCoV S protein or S protein subunit, wherein thelipid nanoparticle comprises 20-60 mol % ionizable cationic lipid, 5-25mol % neutral lipid, 25-55 mol % cholesterol, and 0.5-15 mol %PEG-modified lipid.
 18. The method of claim 17, wherein the open readingframe encodes a BetaCoV S protein.
 19. The method of claim 18, whereinthe ionizable cationic lipid is Compound 25, the neutral lipid is DSPC,and the PEG-modified lipid is PEG-DMG.
 20. The method of claim 18,wherein at least 80% of the uracil in the open reading frame of the mRNAhas a 1-methylpseudouridine modification.
 21. The method of claim 20,wherein the ionizable cationic lipid is Compound 25, the neutral lipidis DSPC, and the PEG-modified lipid is PEG-DMG.